THE PENNSYLVANIA STATE UNIVERSITY

SCHREYER HONORS COLLEGE

DEPARTMENT OF CHEMICAL ENGINEERING

DEVELOPMENT OF AN INDUCIBLE SUICIDE VECTOR FOR THE CONTROL OF

AGROBACTERIUM TUMEFACIENS

NATHANIEL K HAMAKER

SPRING 2016

A thesis

submitted in partial fulfillment

of the requirements

for baccalaureate degrees

in Chemical Engineering and Biology

with honors in Chemical Engineering

Reviewed and approved* by the following:

Wayne R. Curtis

Professor of Chemical Engineering

Thesis Supervisor

Honors Adviser

Phillip E. Savage

Professor and Head of Chemical Engineering

Faculty Reader

* Signatures are on file in the Schreyer Honors College.

i

ABSTRACT

Plants can be genetically manipulated to produce proteins or metabolites of interest following

either transient DNA delivery or stable genetic transformation. Agrobacterium tumefaciens provides a

biologically mediated means of DNA delivery that is both highly effective and potentially scalable in

bioreactor systems. However, the requirement of bacterial co-cultivation with cultured plant tissues

requires subsequent elimination of the bacteria—typically using expensive antibiotics (e.g. moxalactam

and cefotaxime). This requirement for post-transformation removal of Agrobacterium constrains both

scaling and prolongs the process overall (as well as false positive screening for transgenes if the bacteria

persists). The focus of this work was to develop cost-effective mechanisms for facilitating bacterial

eradication. The goal was to demonstrate the feasibility of a synthetic plasmid, harboring an inducible kill

switch to allow for controllable elimination of the bacteria following Agrobacterium-mediated

transformation of plant tissue culture. More specifically, this thesis describes the construction of a lethal

DNA construct harboring a toxic operon that was pieced together from genetic elements sourced from

bacterial toxins, inducible promoters and the T7 bacteriophage expression system. Transcriptional barriers

were included to suppress basal levels of the expression system to prevent premature death due to lax

expression. Endogenous toxins are only produced when triggered by specific chemical signals, designed

as a cost-effective alternative to the aforementioned antibiotics. The construct is intended for eventual

insertion into the genome of the bacteria to create a stable strain with an inducible kill switch. As

transgenic proteins and other plant-derived products have a wide range of lucrative applications in the

agriculture and pharmaceutical industries, such a strain represents a highly desirable asset to any company

involved in plant genetic improvement. This should be particularly useful for the next generation of plants

modified by precise genome editing tools.

ii

TABLE OF CONTENTS

LIST OF FIGURES .................................................................................................... iii

LIST OF TABLES ....................................................................................................... iv

ACKNOWLEDGEMENTS ......................................................................................... v

Chapter 1 : Background Information ......................................................................... 1

Chapter 2 : Introduction ............................................................................................. 6

Auxotrophic Strain Generation ........................................................................................ 6 Suicide Switch .................................................................................................................. 8 Genetic Building Blocks .................................................................................................. 8

Plasmid Stability and Replication ............................................................................ 9 Inducible Promoters ................................................................................................. 10 Bacteriophage T7 RNA Polymerase Directed Expression ....................................... 14

Finding Counterselective Agents ..................................................................................... 17 GhoT ........................................................................................................................ 19 Barnase ..................................................................................................................... 20 Tse1 (Tae1) .............................................................................................................. 21

Kill Switches in Action .................................................................................................... 22

Chapter 3 : Methods ................................................................................................... 24

Assembly of pSU-GhoT ................................................................................................... 24 Growth Inhibition Experiments with pSU-GhoT ............................................................. 28 Assembly of pSU-empty .................................................................................................. 29 Assembly of pTara1300 ................................................................................................... 30 Minimizing the pVS1 Replicon ....................................................................................... 31 Assembly of pSU16R and pSU16F .................................................................................. 32 Preparation of Toxins for Insertion .................................................................................. 36

Chapter 4 : Results and Discussion ............................................................................ 37

Selection Pressure against Lethal Constructs ................................................................... 37 Tightening Expression Control ........................................................................................ 43 Vector Versatility ............................................................................................................. 44

Chapter 5 : Future Work ............................................................................................ 47

Appendix A List of Primers ........................................................................................ 51

Appendix B Additional Plasmid Maps ....................................................................... 53

iii

Appendix C Cloning Strategy Maps ........................................................................... 56

pCR4-lacI-T7RNAP ......................................................................................................... 56 pSU-GhoT ........................................................................................................................ 57 pSU-empty ....................................................................................................................... 58 pTara1300 ........................................................................................................................ 59 pSU16............................................................................................................................... 60 pSU13R ............................................................................................................................ 61 pSU16R ............................................................................................................................ 62 pSU16F ............................................................................................................................ 63

Appendix D Gibson Assembly Details ....................................................................... 64

Appendix E Gibson Assembly: Design and Execution .............................................. 65

Appendix F Media and Reagent Recipes .................................................................... 75

Gibson Assembly Master Mix ......................................................................................... 75 Minimal Defined Media (M9) .......................................................................................... 76

Appendix G GhoT Topology ...................................................................................... 77

Appendix H Sequence of pSU16R ............................................................................. 78

Appendix I Sequence of pSU13R ............................................................................... 80

Appendix J Gel Electrophoresis Tips ......................................................................... 81

BIBLIOGRAPHY ........................................................................................................ 82

Academic Vitae ............................................................................................................ 91

iv

LIST OF FIGURES

Figure 1. Map of WT pTiBo542. ............................................................................................. 3

Figure 2. Diagram of the native lac operon,showing lacI, Plac, lacO and lacZ in series. ......... 12

Figure 3. Structure of the ara regulatory region. Protein binding sites are denoted in PBAD. .. 13

Figure 4. A map of pTara. This binary plasmid has demonstrated the use of T7RNAP-directed

expression under arabinose-inducible conditions. ........................................................... 16

Figure 5. A map of pLZ2-LacY. Unintentionally, the use of this plasmid showed that T7RNAP

can be encoded on the same plasmid as PT7 to express a target. Note that T7RNAP is

oriented in the antisense strain relative to PT7. ................................................................. 17

Figure 6. Map of pET28b-GhoT, used to express the GhoT toxin when a source of T7RNAP is

provided. .......................................................................................................................... 19

Figure 7. The pMT1002 vector is used to express high levels of the potent barnase effector. 20

Figure 8. Expression of this vector causes a suicidal response in E. coli as Tse1 is directed to the

periplasmic space. ............................................................................................................ 22

Figure 9. Map of the commonly used binary vector—pCAMBIA1300 .................................. 25

Figure 10. Cloning vector containing lacI-T7RNAP, isolated from the BL21 genome. ......... 26

Figure 11. Map of pSU-GhoT, the desired assembly product for IPTG-inducible GhoT

expression. ........................................................................................................................ 27

Figure 12. Empty T7 expression cassette designed for versatile gene insertion. Unique RE sites

are shown in bold. ............................................................................................................ 30

Figure 13. The pTara1300 vector was created from pTara500 to eliminate unwanted RE sites. 31

Figure 14. The nonessential region (gray) was removed in subsequent versions of the suicide

vector. ............................................................................................................................... 32

Figure 15. Map of pSU13-trunc. Note the MfeI and XhoI restriction sites for correction of the

araC-T7RNAP deletion ................................................................................................... 33

Figure 16. Map of pSU16R. Orientation of PT7 is flipped relative to pSU16F. ....................... 35

Figure 17. Map of pSU16F. ..................................................................................................... 35

Figure 18. Alignment of mutations in pSU-GhoT(4) and pSU-GhoT(7) against expected sequence

(top). ................................................................................................................................. 38

Figure 19. GhoT expression using pSU-GhoT(7) in E. coli. Induction occurred at time zero. 39

v

Figure 20. Culture comparison of induced (left) versus un-induced (right). ........................... 39

Figure 21. Cell morphology differences between induced (left) versus un-induced (right) at 1000x

magnification. .................................................................................................................. 40

Figure 22. GhoT expression using pSU-GhoT(7) in A. rhizogenes. Induction time is denoted by

▼. ..................................................................................................................................... 41

Figure 23. Dilution plating shows a slight decrease in viability when expression of GhoT is

induced. ............................................................................................................................ 41

Figure 24. Anti-his tag immunoblotting of induced A. rhizogenes whole cell lysate. Although a

negative control was not run, this is the expected size of GhoT+6xhis. .......................... 42

Figure 25. GhoT expression using pSU-GhoT(7) in A. tumefaciens. No difference in growth. 42

Figure 26. Multiple cloning site of pSU16R. After inserting, a gene of interest after PT7, this

vector could be used to express the target protein at high levels in a variety of hosts. .... 46

Figure 27. Map of pSU16R-Barnase........................................................................................ 47

Figure 28. Map of pSU16R-Tse1 ............................................................................................. 48

Figure 29. Map of pSU-MCS ................................................................................................... 53

Figure 30. Map of pSU-empty. ................................................................................................ 53

Figure 31. Map of pSU-trunc ................................................................................................... 54

Figure 32. Map of pTara500. ................................................................................................... 54

Figure 33. Map of pSU16 ........................................................................................................ 55

Figure 34. Map of pSU13R. ..................................................................................................... 55

vi

LIST OF TABLES

Table 1. Relevant Primer and Oligonucleotide Sequences ..................................................... 51

vii

ACKNOWLEDGEMENTS

I would like to thank Professor W. Curtis for his abundance of brilliant ideas and guidance in this

work. He went above and beyond as an advisor by always making himself available for scientific

discussions and troubleshooting. Also, I appreciate Professor P. Savage for his willingness to be my thesis

reader and committee member. The training and concept generation of Dr. Sergio Florez was critical in

accomplishing this work. As my mentor, I am indebted to him for introducing me to the world of

scientific research. I extend my gratitude to Tina Shing Li Lai for her help with protein isolation and

immunoblotting. I am very glad we had the fortune of sharing both an office and a love for plants.

Chandler Thomas and Morgan Shires directly contributed to this work by assisting in plasmid preparation

and running numerous PCRs. They were a pleasure to mentor, and I am truly grateful for their eagerness

to help. I greatly appreciate the support of Erica Lennox in keeping the lab organized and well-stocked, as

well as being available to listen to my scientific musings. Furthermore, I would like to thank Elicia

Yoffee, Krishnan Sreenivas, Rachel Erwin, Ryan Jones and Tucker Langseth for making Curtis lab such a

cheery and enjoyable place to work.

The Penn State Chemical Engineering Department was instrumental in providing materials and

administrative assistance. Additionally, I extend my thanks to Dr. Joseph Mougous and Professor T.

Wood for providing requested plasmids from their collections.

In closing, I owe a debt of gratitude to my friends and family for their frequent encouragement. In

specific, the love and motivation offered by my mother and father was a true blessing. Lastly, I am

indebted to my amazing girlfriend JoAnna for her dedication and support. Even after unsuccessful

experiments and sleepless nights in lab, she was my greatest source of inspiration.

1

Chapter 1 :

Background Information

The gram-negative, soil-dwelling bacteria of the Agrobacterium genus are characterized by their

shared ecological role as plant pathogens1. Depending on the strain, they are specialized to infect certain

organs of the plant, chemotactically driven by metabolites released from epidermal wounds. Not only do

these biochemical signals direct bacterial movement, but also weaponize the cell through upregulation of

virulence genes involved in assembling the protein complexes used in the infection process. Of the

compounds that activate the pathogenic response, acetosyringone, a ubiquitous plant phenolic, is the most

widely understood2; still, some of the simple sugar byproducts of cell wall breakdown have also been

implicated in playing a role3. Remarkably, inducers, like acetosyringone and other similar compounds,

can be artificially supplemented during infection to enhance the response even with gymnosperms and

monocotyledonous plants that do not typically serve as a natural host4,5.

The most notable species of this genus are A. tumefaciens (Rhizobium radiobacter) and A.

rhizogenes, both possessing the extraordinary ability to induce tumor formation via gene transfer to the

plant host. The normal manifestation of these tumors are classified according to affected tissue-type as

crown gall and hairy root for A. tumefaciens and A. rhizogenes, respectively. The genes that are

horizontally transferred from bacteria to plants are carried on 10-25 kilobase fragments called the transfer

(T)-DNA, which is randomly integrated into the genome of the host plant. In wildtype (WT) bacteria,

both the T-DNA and all necessary virulence genes to facilitate DNA transfer and intercellular attachment

are harbored on the tumor-inducing plasmid (pTi or pRi). Bacteria must be cultured at temperatures below

28°C in order to maintain this plasmid; otherwise, the strain becomes avirulent.

Agrobacterium tumefaciens C58 (Accession Nos. AE007869, AE007870, AE007871, AE007872)

was the first strain to have its genome fully sequenced and assembled6,7. Regarding its native T-DNA, the

2

transferred genes consist of tumor-inducing enzymes that synthesize the auxin-class phytohormone indole

acetic acid (IAA), as well as genes for the synthesis of nopaline, an opine. Opines represent a diverse

family of compounds formed from the condensation of an amino acid and a keto acid or sugar moiety;

each Agrobacterium strain is categorized by the specific opine that it causes the plant to produce.

Cleverly, the plant is genetically hijacked into producing this unique carbon and nitrogen source that only

the bacteria is capable of metabolizing while it proliferates in the safety of the hormone-induced gall.

The development of Agrobacterium into a suitable vector for the introduction of genetic material

was a major breakthrough for plant biotechnology. The key to harnessing the bacteria was the decoupling

of the T-DNA from the Ti plasmid. Although many strains with various modified Ti plasmids are

currently available for use, the AGL1, GV3101, MP90, and EHA105 strains remain the most widely used

for plant applications8. Despite being the first created, EHA105 and its predecessor EHA101, remain

popular among plant biotechnology researchers. These strains were named for Dr. Elizabeth Hood, who

created these strains while a Ph.D. student in the lab of Dr. Mary-Dell Chilton, renowned for generating

the first transgenic plant in 19839. EHA101 consists of an A. tumefaciens C58 chromosomal background

harboring a modified version of pTiBo542 (Figure 1). In its native state (NC_010929), this Ti plasmid

carries a gene that confers hypervirulence, as well as two distinct fragments of T-DNA10. For EHA101,

both T-DNA regions were removed and replaced through a double cross-over event with a kanamycin

selection marker11. EHA105 was subsequently created from EHA101 through a similar method to replace

the selection marker with that of tetracycline for the convenience of using kanamycin resistant binary

plasmids12. By reincorporating a functional T-DNA as desired, Agrobacterium can be used as a tool for

delivering heterologous DNA to the nucleus of a plant cell.

3

Figure 1. Map of WT pTiBo542.The virulence (orange) and opine catabolism (purple) gene clusters are

shown. Within the region deleted (black) to generate the EHA strains, lies the two T-DNA (green) segments.

The power of this technique is derived from the use of a binary vector—by placing the T-DNA

onto a separate smaller plasmid, it can be easily edited so that genetic elements of interest (along with a

selectable marker) are placed between the left and right borders of the synthetic T-DNA. Typically, these

manipulations are performed within an intermediate E. coli host. Once desired genes have been inserted,

the construct is transformed into Agrobacterium, which is subsequently used to transfect a plant. A brief

description of this procedure, referred to as Agrobacterium-mediated transformation, demonstrates its

ease and utility: After an initial wash of the plant tissue with a solution of bacteria, the infection process

continues for roughly one week while the two organisms are cultured together on selection-free media. At

this point, the bacteria must be eliminated with moxalactam, cefotaxime or similar antibiotics while plant

transformants are subjected to the appropriate selection agent. Untransformed tissues die off, whereas

4

cells that harbor the T-DNA survive and can be propagated as a transgenic cell line or regenerated by

somatic embryogenesis and/or organogenesis into a complete transgenic plant.

Certainly, other methods exist for the introduction of foreign DNA into plants, including

protoplast electroporation, biolistics, microinjection and viral delivery, among others. Depending on

factors such as species, cell type and application, one of these may be preferred. Still, the Agrobacterium-

mediated transformation remains popular due to its versatility and high efficiency. It also shines as the

most effective method for the stable integration of DNA into the host genome, whereas many of the other

non-biological approaches terminate with transient expression events13. Even in cases where transient

expression is desired, as the phenomenon is usually characterized by a rapid burst of protein expression,

certain Agrobacterium strains have been optimized for this purpose14. In fact, the scope of this tool

extends beyond plants as certain derivatives of the bacteria have proven effective in transfecting

unicellular algae or even fungal cell lines with comparable efficiency15. In these cases, protocol

modifications are necessary; still, Agrobacterium-mediated transformation of certain filamentous fungi

cultures have shown a 600-fold improvement in transformation efficiency over conventional methods16.

Given its wide range of utility, continued procedural optimizations and strain engineering is likely to lend

itself to additional beneficial applications.

Focusing on plants, there is a vast and exciting potential that awaits in the creation of transgenic

lines. Apart from enhanced crops that reduce the need for applied pesticides and fertilizers, plants can be

genetically engineered for the synthesis of biological therapeutics and valuable metabolites. Plant-based

platforms for the expression of medicinal protein products, like monoclonal antibodies, have shown to be

a viable contender in pharmaceutical manufacturing using tobacco lines that mimic mammalian

glycosylation patterns17. Subjected to significantly less strict regulations, the production of veterinary

immunotherapies using plant-based platforms has shown to be efficient and low-cost18. Plants cost

relatively little to propagate as all primary and secondary metabolites are synthesized de novo using just

carbon dioxide and sunlight as carbon and energy inputs, respectively. Because they come equipped with

5

a vast array of unique metabolisms, plants can be readily harnessed as chemical factories for unique

metabolites. For example, genetic enhancement of existing pathways, such as the synthesis of paclitaxel,

colchicine and vitamins, would improve yields and cut production costs. Additionally, plants do not act as

carriers for agents of zoonotic diseases, reducing contamination risk and increasing product safety. As one

might guess, there is great motivation to perfect the technologies involved in transfecting and mass-

producing large batches of transgenic plants.

The most promising method due to its scalability and capacity for sophisticated process control is

the use of bioreactors. Developing such a scaled system for rapid transient expression in bioreactors has

been a long term goal of the Curtis lab and includes patents on suspension culture transient expression19,

as well as low cost plastic-lined bioreactor systems that were demonstrated for this specific goal20.

Additionally, the Curtis lab has been developing propagation bioreactors for differentiated plant tissues21,

where there has been the goal of utilizing the delivery of DNA by Agrobacterium as the basis of

manipulating the genetic environment of plant tissues for the induction of somatic embryos. In any liquid-

containing system, mass-transfection would likely be facile upon injection of Agrobacterium yet severely

limited by the need to subsequently eliminate the unwanted bacteria before they can outgrow the plant

cells. Currently, unreasonable amounts of expensive antibiotics would be needed to quickly purge the

reactor of the contamination.

6

Chapter 2 :

Introduction

To accomplish the intended goal of developing a stringent genetic mechanism for the controlled

extermination of Agrobacterium, a previously employed method was evaluated. Ideally, the selected

approach would improve upon past efforts in terms of economic feasibility, process viability, and

suitability for plant cell culture applications. Appropriately, the research group of Dr. Wayne Curtis is

responsible for the earlier advancement in question: the generation of auxotrophic Agrobacterium strains.

Auxotrophic Strain Generation

Auxotrophy is the inability of an organism to synthesize a metabolite necessary for survival;

therefore, the absent chemical (or an appropriate precursor) must be supplemented to the culture media in

order to restore growth and development. Auxotrophs are generated through the disruption of the native

metabolic pathways via mutagenesis techniques. Transposon insertion mutagenesis using suicide vectors

has been very effective historically for its capacity to carry a selectable marker and inability to revert to

WT in the absence of the transposase22. Following mutagenesis, bacteria can be selected for the desired

auxotrophic phenotype by replica plating onto plates deficient in certain metabolites. Of note, the advent

of targeted genome editing techniques, such as CRISPR-Cas9 and TALEN, has streamlined the process of

genetic engineering without having to depend on random insertional events; this applies when the gene of

interest or genome of the organism has been sequenced.

Even under supplemented growth conditions, normal growth cannot always be entirely restored,

leaving the bacteria in a relatively weakened state. In the case of strains used in Agrobacterium-mediated

transformation, this debilitation is preferable as the usual bacterial growth rate is 20 times that of plant

cells, leading to overgrowth23. Of significance to concerns regarding the biosafety and containment of

genetically engineered organisms, escaped auxotrophic bacteria present a lower risk as they are unlikely

7

to survive in the absence of human intervention. On the other hand, infectivity can be adversely affected

as was shown for Agrobacterium mutants auxotrophic for methionine, adenine or asparagine24. Naturally,

this loss of infectivity translates to a drop in plant transformation efficiency because T-DNA is no longer

effectively delivered to the host23. Clearly, it is important that these traits remain balanced in order to

create a useful strain.

Similar to other bacteria in the Rhizobiales order, WT Agrobacterium is capable of synthesizing

its nucleotides and amino acids de novo, providing the opportunity to generate a diverse array of

auxotrophic mutants. For the purpose of preventing overgrowth in liquid co-cultures during induction of

transient expression, auxotrophic mutants of A. tumefaciens EHA105 were generated23. Although this

strain already harbors a tetracycline marker12, the insertional transposons that were used in mutagenesis

also carried a tetracycline marker and required selection using higher antibiotic doses. This was done in

order to facilitate the use of common binary plasmids. Remarkably, one of the mutants generated in this

study auxotrophic for cysteine (designated cys-32) demonstrated an 85-fold enhancement in transient

protein expression compared to the WT A. tumefaciens response.

Auxotrophic mutants have proven to be capable of maintaining high levels of protein expression

while reducing overgrowth. As consequence, they represent a valuable tool in large scale plant co-

cultures. The bacteria must still be subsequently eliminated from the system; otherwise, bacteria would

persist indefinitely by acquiring nutrients from the plant cells. In manufacturing situations involving the

production of human therapeutics, bacterial persistence would also be a major complication with respect

to clearance regulations. Overall, less antibiotics would likely be needed, which would reduce stress on

the plant cells. Still, some amount of expensive antibiotics would still be required, which poses an

economic limitation to scale up. For this reason, an alternative approach was taken: construction of a

lethal genetic kill switch under the control of a low-cost inducer.

8

Suicide Switch

The utility of a genetic kill switch lies in its capacity for induction when the appropriate ligand or

substrate is added. In the “off” state, the lethal agent must remain unexpressed or inactive. The “on” state

is lethal through the activation of a counterselective agent, typically a gene that causes death in the cells

that harbor it. For this system to be effective, the difference between the “on” and “off” state must be

sufficiently strict to prevent unwanted selection pressure, yet sensitive and potent enough to eliminate

virtually all of the bacteria.

Once complete, the lethal cassette would be intended for integration into the bacterial genome (Ti

plasmid or main chromosome) or harbored on an inconspicuous ternary plasmid. For the purpose of

testing potential kill switches, containing varying counterselective markers, it would be useful to develop

these constructs on a high-copy number, broad host range plasmid. This would allow for expedient

cloning and testing in both E. coli and Agrobacterium spp. Preferably, each element would be flanked by

unique restriction enzyme sites to facilitate substitution when optimizing the promoter/counterselection

combination. A variety of genetic building blocks and cloning techniques were utilized to generate these

suicide plasmids.

Genetic Building Blocks

This work is meant to provide an outline of the steps taken to design and construct an inducible

counterselection cassette for use in Agrobacterium strain engineering. In order to build a minimal vector

that carries an effective kill switch, it is necessary to provide an overview of all relevant genetic elements

to consider.

9

Plasmid Stability and Replication

Each plasmid must have one or more vegetative origins of replication (oriV) in order to replicate

and maintain stability in the host cell line; it is also used to control the plasmid copy number per cell. The

oriV is an AT-rich region of the plasmid, essential for the binding of its respective replication protein (e.g.

TrfA, RepA, pBBR1 Rep, and Pir)25,26,27,28. Alternatively, a ColE1/pMB1-derived origin is quite common

in most basic cloning vectors due to its high-copy number (500-700), but relies strictly on E. coli

chromosomal genes to regulate RNA priming29,30. In cases where two plasmids with the same oriV exist

within the same host cell, incompatibility will usually occur when both require the same protein

machinery for replication. In this manner, incompatibility groups have been designated, which can be

exploited to cure a strain of its plasmid(s). On the other hand, it is critical to ensure the compatibility of

plasmids when more than one will be used in the strain as is the case with Agrobacterium binary vectors.

When a plasmid carries its own replication genes and/or is able to employ the machinery of a variety of

diverse microbes, it is considered a broad host range vector. In designing the ideal suicide vector, it

became apparent that the plasmid would need to be capable of replication in both Agrobacterium spp. and

E. coli.

Besides replication, several other regulatory elements are responsible for plasmid stability and

mobilization. Genes, such as staA, incC, sopA and parA, confer segregational stability by ensuring proper

partitioning of the plasmid during cellular fission26. Remarkably, this system is so effective in plasmids

carrying the minimal pVS1 replicon, including most common Agrobacterium binary vectors, that they can

be stably maintained even in the absence of selection pressure26,31. Similar to the oriV, the origin of

transfer/basis of mobility, typically designated oriT/bom, acts as a binding region for its respective

transfer protein during conjugation. This allows for plasmid transfer to an intended host through mating in

the presence of another strain harboring the proper conjugational machinery (tra operon); specifically,

plasmids harboring the ColE1 oriT are mobilized through triparental mating with a pRK2013 E. coli

helper strain26,31,32. In cases where efficient transformation protocols, like freeze-thaw and electroporation,

10

have been established for the desired host (as is the case with Agrobacterium), conjugation is usually

unnecessary. Still, the presence of an oriT can significantly expand the host range of a vector to a wide

variety of gram-negative bacteria32.

Inducible Promoters

A reasonable approach to building the kill switch mechanism would be the use of an inducible

promoter, which provides a way to activate expression when desired. An array of native bacterial

promoters, with those of E. coli being the most studied, exhibit this characteristic. Evolution has done an

impressive job in generating natural genetic systems that decrease wasteful gene expression in cases

where the protein product is only useful under certain conditions. In many bacteria, regulators are of the

lacI or araC families, containing DNA binding domains specific to operators of a nearby gene or

operon33,34. In cases of negative regulation, repression is typically relaxed by the presence of a metabolic

substrate of the gene(s) being regulated. The protein remains bound at the operator, shielding the

promoter and preventing RNA polymerase recruitment, until an inducer binds to an allosteric site. This

triggers a conformational change, releasing the repressor and allowing transcription of the regulon. A

variety of these systems have been characterized, and one must carefully consider the level of repression

and method of induction when selecting an appropriate promoter system. For example, the

thermosensitive ʎ phage cI587 repressor can be used to strictly regulate gene expression at low

temperatures; induction occurs when the culture is raised to 42°C, causing the repressor complex to

unbind from the DNA35. In fact, basal expression levels in the “off” state are sufficiently low even for the

purpose of expressing toxic genes36,37. However, this temperature shift would be severely damaging to

plant cells because activation is intended to occur during the Agrobacterium co-culture. The energy

requirement to heat the system would also pose a challenge when working with large culture volumes.

The rhamnose operon repressor has also proven to be quite effective in the controlled expression of high

11

levels of toxic proteins38. However, at 2 kb, the length of the control element is about twice as long as

other prospective repressors. Posing an even greater challenge, this sequence contains a large number of

useful restriction enzyme sites, limiting the multiple cloning site. For these reasons, other inducible

systems were preferable for the task at hand.

The E. coli repressors lacI and araC, for which their gene families were named, have been

thoroughly characterized and tested in various hosts. Each acts as the gatekeeper of its corresponding

regulon. The function of the lacZYA and araBAD operons is to encode enzymes that catabolize lactose

and L-arabinose, respectively39. Thus, it is not surprising that these metabolites act as inducers to activate

transcription. Still, regulation of each of these operons is somewhat more complex so both will be

explored in greater detail.

As the most studied and well-understood genetic regulatory mechanism known, the lac operon of

E. coli (Accession No. J01636) is the classic example for demonstrating transcription regulation.

Upstream of lacZYA, lacI is under the control of a constitutive promoter. In its resting state in the absence

of lactose, the lacI repressor protein remains bound to an operator region lacO at the promoter Plac,

blocking RNA polymerase (RNAP) and preventing expression of the operon (Figure 2)39. When lactose

is present, it allosterically inactivates the DNA binding of lacI, but the operon still needs a secondary

factor before transcription can occur. Referred to as catabolite repression, the presence of glucose plays a

major role in controlling the lac regulon. When glucose levels are elevated in the cell, a kinase inactivates

both lactose permease and adenylyl cyclase, reducing lactose uptake and decreasing levels of cyclic AMP

(cAMP)40. A catabolite activator protein (CAP) binding site lies within the sequence of Plac, which aids in

the recruitment of RNAP when CAP is present—this DNA binding can only occur when CAP is bound to

cAMP. Therefore, the switch remains “off” if glucose is present even when lactose is also available.

Alternatively, the “on” state occurs only when lactose is available and glucose is absent.

12

Figure 2. Diagram of the native lac operon,showing lacI, Plac, lacO and lacZ in series.

Before moving on, it is important to note that mutant forms of the canonical lac elements come in

a wide variety and are common features of many basic cloning vectors. The lacUV5 promoter is an

upregulated form of Plac, wherein expression is significantly increased due to enhanced recruitment of

RNAP to the site of transcription initiation41. This promoter form is also free from catabolite repression

due to inactivation of the CAP binding site. Modified versions of other lac elements have also shown

enhanced function and improved repression, which will be discussed in Chapter 4.

The ara operon of E. coli is controlled by a mechanism similar to that of lac in that catabolite

repression is facilitated by a CAP binding site, and the araC regulator is affected by L-arabinose

induction. On the other hand, the araC gene regulates its own expression, and its protein product is

responsible for both negative and positive regulation of the ara operon (Figure 3). In the absence of

arabinose, an araC homodimer remains bound to two separate operators. This causes the DNA to form a

loop, which restricts access to the araC promoter (PC), araBAD promoter (PBAD) and CAP. In the absence

of glucose, transcription can occur when two molecules of L-arabinose bind to the allosteric pockets on

each of the araC monomers, moving one of the monomers onto an activating operator site. Because this

also activates expression of araC, the shielding loop will be reformed once the arabinose has been

depleted. Schleif provides an excellent depiction of this mechanism in his review of AraC42. Overall, the

conditions for the “on” state are high arabinose and low glucose.

13

Figure 3. Structure of the ara regulatory region. Protein binding sites are denoted in PBAD.

Although somewhat complicated, the details of the lac and ara mechanisms are nontrivial to the

design of an Agrobacterium kill switch. These systems, native to E. coli, are not conserved in

Agrobacterium so it was relevant to determine whether the machinery would be compatible. One potential

concern was that Agrobacterium RNAP might not be readily recruited to the E. coli promoters. Because

the two types of bacteria prefer differing carbon sources, it was unclear whether similar mechanisms of

catabolite repression and CAP binding would translate. Another concern was whether Agrobacterium

would be capable of facilitating the import of inducers into the cytoplasm. In the case of using an

arabinose inducible switch, this sugar has been implicated in the virulence and chemotaxis response of

Agrobacterium by activating expression of the chv operon43,44. Fortunately, much insight was gained from

the work of research groups using broad host range expression vectors in A. tumefaciens. Chen et al.

showed that when a Plac plasmid lacking a copy of lacI was used, basal and induced expression were

equally high; expression levels were lower in E.coli when using the same plasmid (due to the endogenous

copy of lacI). After lacI was added, β-galactosidase assays indicated very low basal expression and

moderate levels when induced45. Newman et al. compared the same lacI-Plac plasmid to a PBAD plasmid in

A. tumefaciens. Induced expression was lower with PBAD by roughly 12-fold at the same concentration of

inducer (1 mM); Plac basal expression was only slightly lower than that of PBAD. By measuring expression

in the presence of various primary metabolites, A. tumefaciens was shown to be most sensitive to

catabolite repression in the presence of TCA cycle intermediates, particularly malic acid with a 94%

reduction in expression. Of practical concern, sucrose, which is a common ingredient in plant cell culture

14

medias, decreased PBAD expression levels by 56%46. The same catabolite repression studies were not

performed with Plac but would be expected to give similar results.

The capacity for strictly repressed, yet inducible, expression is essential when attempting to

produce a lethal target protein. Both Plac and PBAD represent tightly controlled regulatory elements in A.

tumefaciens sensitive to low levels of inducer (1 mM). Of note, isopropyl β-D-1-thiogalactopyranoside

(IPTG) is used in place of lactose in lacI-Plac expression systems. This compound is not sensitive to the

presence of lactose permease and cannot be catabolized; therefore, IPTG levels remain constant during

induction and much less is required.

Bacteriophage T7 RNA Polymerase Directed Expression

To achieve the highest levels of protein expression in bacterial platforms, native bacterial-derived

promoters are suboptimal. Alternatively, bacteriophages, which typically have no regard for conserving

the resources and protein machinery of their host, deliver unmatched protein expression. By exploiting

certain phage-derived elements, it is possible to direct maximal expression of desired genes.

Bacteriophage T7 (Accession No. NC_001604) has been exploited for this purpose, spawning the

ubiquitous pET vectors for use in E. coli. This phage possesses its own unique RNA polymerase (gene 1;

T7RNAP), capable of overpowering the machinery of its E. coli host with substantial transcription levels.

T7RNAP recognizes unique promoter sequences found only within its own genome and not within the

host to rapidly replicate47. After phage particles have assembled, the host is lysed by T7 lysozyme, and

the virion moves on to its next victim.

To utilize this effective system, the T7RNAP was carefully isolated so that the fragment did not

also carry a copy of the T7 promoter (PT7). In the T7 phage genome, T7RNAP is followed by several

copies of PT7; if these were to be moved together onto a circular vector, the T7RNAP would likely begin

to auto-induce its own expression as it transcribed around the length of the plasmid. Even in the absence

15

of any phage toxins, this load on the cell would halt normal cell growth. Davanloo et al. experienced this

challenge as the first to clone the gene48. After its eventual isolation, T7RNAP was put under the control

of lacI-lacUV5. The construct was inserted into the genome of E. coli by ʎ phage integration to create

strain BL21(DE3) (Accession No. NC_012892.2). Subsequently, the pET vector series was constructed,

which harbors a T7 expression cassette. This cassette consists of a multiple cloning (MCS) site that is

flanked by PT7 and a T7 terminator; the highly efficient ribosomal binding site (RBS) of T7 gene 10 was

also incorporated to enhance translation49. A lacO sequence was added adjacent to PT7, allowing for

further repression by lacI. In conjunction with a pET vector carrying a target gene, the BL21(DE3) strain

can be used to quickly accumulate the target protein to levels approaching 50% of the total cell protein.

Following IPTG induction, this system completes expression in less than three hours47.

In strains that do not harbor a copy of T7RNAP, protein expression of the T7 expression cassette

is wholly inactive. Conveniently, this allows for plasmid manipulation in the absence of unwanted

selection pressure (expression of an arresting or toxic target protein) that may lead to mutations of the

desired gene. The standard IPTG-dependent system delivers an all-or-none response, shifting from basal

expression to maximal expression without much room for modulation. This inflexibility is of little

consequence when high yields of the target are desired, provided that the culture is given time to grow to

a sufficient density before induction. To improve versatility and enhance repression, Wycuff et al.

constructed a binary vector designated pTara (Figure 4), which would act as the chromosomal source of

T7RNAP under the control of araC-PBAD. In conjunction with a pET plasmid vector, this system was

shown to moderate expression levels using competing amounts of glucose and arabinose50. Subsequently,

pTara500, a pTara derivative with an enhanced RBS of the T7RNAP transcript, was also added to the

library of useful tools for protein expression51.

16

Figure 4. A map of pTara. This binary plasmid has demonstrated the use of T7RNAP-directed expression

under arabinose-inducible conditions.

Because of its high, rapid expression, this system would be of use to an Agrobacterium kill switch

to ensure a potent response after induction of the counterselection gene. Alone, the lacI-Plac plasmid of

Chen et al. was unable to reach maximal expression in A. tumefaciens after 16 hrs of induction45. By

placing T7RNAP under the control of even a weak promoter, any response would be considerably

amplified. Also, the high specificity of PT7 for its corresponding T7RNAP should prevent expression until

bacterial elimination is desired (because native RNAP will not transcribe at PT7). Kang et al. built a vector

(Accession No. EF153732) carrying a transposon with the genetic elements necessary for IPTG-inducible

T7RNAP expression similar to that of E. coli BL21(DE3). The transposon can be inserted into the

genomes of a wide range of hosts. In fact, A. tumefaciens was engineered with this system and

subsequently used to demonstrate inducible expression of a target protein under PT752. So far, the T7

expression systems described have used genetic elements that are spatially separated in the host; typically,

T7RNAP is expressed from the chromosome, while the T7 expression cassette is harbored on a plasmid.

17

Pertinent to this work, Zhu et al. used pLZ2-LacY (Figure 5; Accession No. KJ641600) to show that all

necessary elements for T7 expression of a target protein can be carried on the same vector53.

Figure 5. A map of pLZ2-LacY. Unintentionally, the use of this plasmid showed that T7RNAP can be

encoded on the same plasmid as PT7 to express a target. Note that T7RNAP is oriented in the antisense

strain relative to PT7.

Finding Counterselective Agents

Lethal genes are ubiquitous in bacteria, due to their role in plasmid stability and bacteriostasis. In

the resting state, the host is rendered immune to the toxin through co-expression of an antitoxin. Toxin-

antitoxin (TA) systems naturally function as a clever genetic tool for maintaining the stability of

transferable genetic elements, like plasmids and mobile elements. In this mechanism, the maintained

element harbors both the toxin and antitoxin genes, which are transcribed as a bicistronic unit to ensure

equal amounts of each. Often, the toxin is a much more stable protein compared to its antitoxin

counterpart, with which it forms an inactive complex. When fission occurs, TA complexes in the

cytoplasm are passed to both daughter cells, whereby growth continues as normal in cells that have also

18

received the TA genetic element. On the other hand, cells that do not receive a copy of the TA element

during segregation are killed when the unstable antitoxin eventually degrades, and the active toxin is

released54. In this manner, genetic elements are maintained even in the absence of external selection

pressure. Although most TA systems abide by this general mechanism, the specific mode of action of the

toxin is usually unique. For example, the ccdB toxin of the native E. coli F plasmid acts as a poison to

DNA gyrase, bringing all DNA replication within the host to a lethal halt55. This gene is now used in a

variety of cloning vectors as a counterselection. Alternatively, pTiC58 of A. tumefaciens is maintained by

the ietAS TA pair, in which the ietS gene encodes a lethal serine protease56. Interestingly, some TA

systems are not wholly lethal when the toxin is active, but instead promote a bacteriostatic response; this

means that bacterial growth is inhibited, but cells persist. These persister cells remain dormant and

unaffected by many external stresses without undergoing genetic change; thus, maintaining the survival of

the culture through devastating events57. Although the vast complexities of TA systems are beyond the

scope of this work, those that offer toxins with the most potent effects are of greatest interest.

Similarly, effector-immunity systems consist of a poison and antidote, but the lethal effects are

directed at other cells. Warfare is often waged amongst bacterial species in order to compete for shared

resources. As an offensive tactic, lethal effector proteins are secreted into the environment or injected

directly into rival cells. Prior to secretion, complementary immunity proteins protect the host from the

adverse effects of the bactericidal effector. Sharing common origins with the toxins of TA systems,

effectors have evolved for the sole purpose of rapidly debilitating unwanted neighbors. Much is still left

to be determined about the inner workings of effector-immunity systems, as well as antagonistic bacterial

interactions in general, but Benz et al. provides an excellent review of current knowledge58. Naturally,

proteinaceous effectors and toxins serve as valuable pools from which to draw lethal protein targets for

inducible expression. An overview of those most relevant to this work will now be provided.

19

GhoT

Recently discovered, GhoT-GhoS represents a toxin-antitoxin pair encoded on the main

chromosome of E. coli. TA systems are characterized by the way in which the antitoxin inactivates its

toxin counterpart; this pair represents the first type V system, whereby the antitoxin (GhoS) cleaves the

mRNA of the toxin (GhoT). In fact, ghoST is transcribed as a bicistronic unit. The GhoT toxin is a

membrane protein with two transmembrane domains and was shown to increase both cell lysis and

persistence; when ghoT is expressed in E. coli, cells exhibit a ghostly transparent morphology due to

membrane damage. When the chromosomal copy of ghoS was deleted, a plasmid expressing ghoT (such

as the one shown in Figure 6) resulted in complete lysis of the culture with no colonies forming on an LB

plate59. Although ectopic expression leads to cell lysis, low levels of endogenous ghoT expression are

solely responsible for reduced metabolism and increased persistence. At this level, the membrane is

somewhat damaged to the point that proton motive force is reduced (reducing cellular ATP), but the

membrane is not wholly destabilized60. Further work is needed to characterize the exact mechanism by

which GhoT causes membrane damage.

Figure 6. Map of pET28b-GhoT, used to express the GhoT toxin when a source of T7RNAP is provided.

20

Barnase

Barnase is a well-characterized ribonuclease effector secreted by Bacillus amyloliquefaciens.

Barstar offers immunity to the potent effector; the inactive complex formed by the two proteins exhibits a

notably high binding strength61. Unlike most effector-immunity and TA systems, the pair is not expressed

as an operon. Early cloning and expression of barnase for purification proved difficult due to its severely

damaging effects on host cells, even under strict promoters; therefore, the pMT1002 expression vector

(Figure 7) was constructed, on which barnase was placed under the control of the thermosensitive ʎ

phage cI587 repressor. The effector was also tagged with a PhoA leader to direct expression to the

periplasmic space where barnase is nonfunctional. Downstream, barstar is under the control of its native

promoter36. E. coli secreting recombinant barnase was shown to inhibit the growth of several species of

co-cultured Rhizobiales, which happen to be closely related to Agrobacterium62. In fact, barnase exhibits

a broad host range biocidal effect and is used as an effective counterselection in both prokaryotes and

eukaryotes63,64.

Figure 7. The pMT1002 vector is used to express high levels of the potent barnase effector.

21

Tse1 (Tae1)

Despite having an arsenal of DNase effectors of its own, A. tumefaciens loses in competition with

Pseudomonas aeruginosa (PA) when co-cultured in LB. In fact, the aggressive counterattack of PA is

triggered by the presence of weaponized A. tumefaciens—the same response is not observed in co-culture

with A. tumefaciens mutants lacking an effector secretion complex65. For this reason, PA was explored

as a source of potential toxins with which to construct an effective kill switch. Russel et al. showed that

PA secretes three proteinaceous effectors Tse1-3 (Type-VI Secretion Export) in tandem, effective against

a wide range of gram-negative rivals. A type VI secretion system (T6SS) is used to translocate these

effectors from the cytoplasm of PA directly into the periplasmic space of adjacent cells, bypassing its own

periplasm in the process; this was the first T6SS found to be discovered with this function66,67. Tse1 and

Tse3 were renamed Tae1 and Tge1, respectively, when their gene families were characterized68. For

consistency, the original names will be used in this work. Both Tse1 and Tse3 are active in the periplasm,

causing catastrophic damage to the peptidoglycan (PG) wall. Specifically, Tse1 functions as an amidase

that cleaves the γ-D-glutamyl-L-meso-diaminopimelic acid amide bond of crosslinked PG. Similarly,

Tse2 is a muramidase that cleaves the PG backbone. Interestingly, Tse2 is lethal in the cytoplasm, but its

mechanism remains to be elucidated69. Each effector has its own respective immunity protein, designated

Tsi1-3. Although Tse1 and Tse3 reside in the cytosol of PA prior to secretion, a reserve of Tsi1 and Tsi3

is kept in the periplasm to counter friendly fire of neighboring PA cells58. Tse1 was selected for use in this

work because it elicited the most rapid kill response when suicidally expressed in E. coli (refer to the

Tse1 expression vector in Figure 8); a PelB leader was used to target the periplasm67. Additionally, it is

the relatively well-characterized with active sites determined and a resolved crystal structure69.

22

Figure 8. Expression of this vector causes a suicidal response in E. coli as Tse1 is directed to the

periplasmic space.

Kill Switches in Action

Using many of the genetic elements described above, a collection of inducible suicide systems

has been developed for the purpose of allelic exchange and biocontainment of transgenic bacteria. The

former application exploits a two-step approach using a vector that is non-replicable in the host; the

vector carries a positive selection that must be integrated (along with the desired construct) into the host

genome by intermolecular crossing over to survive selection. Because the construct also harbors a

counterselection, a second crossing over event must then occur, in which the now lethal remains of vector

DNA are ejected from the host; thereby, forcing exchange with a homologous target sequence. Reyat et

al. provides an excellent review and schematic of this useful mechanism70. Following this scheme, lactose

and arabinose inducible suicide plasmids (Accession Nos. KT326152, KT326153) have been developed

for use in Vibrio spp. using the vmi480 gene product as the induced counterselection. This marker was

23

derived from a TA pair native to Vibrio mimicus VM57371. A variety of other allelic exchange systems

for use in gram-negative bacteria utilize the SacB levansucrase gene of Bacillus subtilis. Unlike

previously described mechanisms, the counterselection can be constitutively expressed without any

adverse effects; thus, eliminating the need for an inducible promoter. Instead, the substrate of this

enzyme, sucrose, undergoes a lethal reaction in the periplasmic space of the bacteria when added to the

culture; this counterselection has demonstrated to be effective in A. tumefaciens72. Other such substrate or

ligand mediated toxic mechanisms exist, such as the system used to generate deletions in the protist

Trypanosoma cruzi (Chagas disease); the ligand triggers a conformational change, converting the toxin

protein into an active form73.

Counterselection methods for biocontainment and controlled cell death operate in a similar

manner, but the bacteria is offered no means by which to escape selection; Endogenous survival

mechanisms that could potentially complicate this effect will be discussed in a later chapter. Conditional

suicide vectors have shown to be somewhat effective in eliminating E. coli strains that harbor a lethal

vector74,75. Use of an E. coli hok (host-killing) gene, as an inducible counterselection, resulted in 90 to

99% cell death. As an undetermined consequence of insensitivity, surviving cells did not grow normally

even when cultured without an inducer74. On the other hand, Recorbet et al. engineered an E. coli strain

harboring the aforementioned sacB gene within the main chromosome. Sucrose induction was highly

effective in lysing cells with an efficiency of 99.9%. When the strain was released into the soil microcosm

and later treated with a dilute sucrose solution (0.5 g L-1), the number of E. coli dropped 10-3-fold76. From

these studies, it is apparent that the method of counterselection and the stringency of the “off” state play a

major role in controlled cell death. Dependent on its mechanism of action, a toxin may not be sufficiently

potent to achieve a desirable killing efficiency. Additionally, cloning of the hok construct continuously

failed until a strain of E.coli with enhanced LacI repressor expression was used74. In light of these

insightful experiments, strategies for building an Agrobacterium kill switch were implemented.

24

Chapter 3 :

Methods

See Appendix C for picture maps of the cloning strategies used to generate each of the vectors

described in the following sections. SnapGene software (from GSL Biotech; available at snapgene.com)

was critically useful for designing and visualizing all DNA sequences described herein. A detailed

protocol for Gibson Assembly is also provided in Appendix E, while more basic molecular cloning

techniques (e.g. restriction enzyme digest, ligation, transformation, etc.) can be found in the manual by

Green and Sambrook77. All colony PCR screening was performed using GoTaq® Green Master Mix

(Promega). Additionally, all primers used are given in Appendix A. Unless specified, proprietary

enzymes were used according to protocols available from their respective distributor (i.e. New England

Biolabs® Inc, Thermo Fisher Scientific Inc, or Promega Corporation). Selection agents (ampicillin,

chloramphenicol, and kanamycin) and most media components were purchased from Sigma-Aldrich

Chemical Co.

Assembly of pSU-GhoT

The first approach at constructing the inducible kill switch was to use the lacI-T7RNAP cassette

from BL21(DE3) to express GhoT, contained on a single vector. Because the construct would eventually

be tested in E. coli and Agrobacterium, a binary vector—pCAMBIA1300 (Figure 9) was selected as the

backbone [provided by the Centre for Application of Molecular Biology for International Agriculture

(CAMBIA), Australia]. Because convenient restriction sites were unavailable and more than two

fragments were being combined, Gibson Assembly was selected as the method to build the construct in

the fewest number of cloning steps.

25

Figure 9. Map of the commonly used binary vector—pCAMBIA1300

BL21(DE3) genomic DNA was extracted using a Wizard® genomic DNA Purification kit

according to manufacturer’s instructions (Promega). Initial attempts to directly amplify the lacI-T7RNAP

cassette from genomic DNA with primer pair A17 & A20, designed with overhangs for Gibson

Assembly, were unsuccessful. Instead, the cassette was amplified using primer pair A30 & A33, without

overhangs, using a touchdown PCR with Phusion® polymerase. The touchdown PCR started with an

annealing temperature of 72°C and decreased to 54°C over 18 cycles before cycling another 20 times at

the final annealing temperature. The PCR product was verified by gel electrophoresis and extracted using

a GeneClean® kit (MP Biomedicals). Because Phusion® polymerase generates blunt-ended products, 3’

A overhangs were added to the fragment before ligating into a pCRTM4-TOPO® vector (Invitrogen). The

ligation product was transformed directly into chemically competent E. coli TOP10 and plated on LB

with ampicillin selection. Resulting transformants were verified by colony PCR, and one colony (4) was

selected for cryostorage. The plasmid was named pCR4-lacI-T7RNAP (Figure 10).

26

Figure 10. Cloning vector containing lacI-T7RNAP, isolated from the BL21 genome.

Touchdown PCR (see description above) was used to amplify the lacI-T7RNAP cassette from

pCR4-lacI-T7RNAP with primer pair A17 & A20. The T7-ghoT expression cassette was amplified with

primer pair A21 & A22 from pET28b-GhoT (Figure 6), a gift from Thomas Wood59. Notably, the ghoT

gene in this cassette includes a 6xhistidine tag on the C-terminus of the protein. Both PCR products were

size verified and subsequently gel purified. The pCAMBIA1300 backbone was linearized with EcoRI; to

prevent self-ligation, 5’ phosphate groups were subsequently removed using Antarctic Phosphatase

(NEB). The three fragments were combined at a femtomole ratio of 40:45:50 (pCAMBIA1300:lacI-

T7RNAP:T7-ghoT) in a 20 µL Gibson Assembly reaction (see Appendix F for master mix recipe). The

reaction was incubated at 50°C for 1 hour and immediately transformed into chemically competent E. coli

TOP10 (prepared by standard methods77). Cells were plated on LB+0.3% glucose with 50 mg/ml

kanamycin selection. Glucose was added to reduce basal expression of the toxin by additional suppression

of the lac operon. 20 colonies were screened by colony PCR and three appeared to test positive for T7-

ghoT cassette. These were re-streaked, and a second round of colony PCR revealed that one of the

27

original three colonies had been a false positive. From this plate, two descendant clones (arbitrarily

designated 4 and 7) of the original true positives were preserved. Plasmids were extracted from each by

the alkaline lysis method and sent for sequencing at the on-campus sequencing center (dnaLIMS).

The desired plasmid product of the aforementioned Gibson Assembly was named pSU-GhoT

(Figure 11). Both pSU-GhoT(4) and pSU-GhoT(7) (according to colony number) showed mutations in

the overlap region between the lacI-T7RNAP and T7-ghoT cassettes. The potential impact of these

mutations was evaluated and will be discussed in a later chapter; pSU-GhoT(7) was selected for growth

inhibition studies. It was discovered that primer A20 was designed incorrectly with an extra nucleotide,

contributing to the mutations observed at the overlap. This primer was corrected when building

subsequent vectors.

Figure 11. Map of pSU-GhoT, the desired assembly product for IPTG-inducible GhoT expression.

28

Growth Inhibition Experiments with pSU-GhoT

First, pSU-GhoT(7) was tested in E. coli TOP10. In this growth study and all subsequent studies

using other bacterial species, LB+0.3% glucose with kanamycin selection was used as the growth

medium. Overnight cultures were grown to an OD600 of ~2. This was diluted in 5 mL to an OD600 of 0.1,

which was split into two culture tubes of 2.5 mL each. Cultures were grown at 37°C on a shaker at 250

RPM. At time zero, one of the tubes was induced with 0.1 mM, and the OD600 was measured regularly for

5 hours by taking 100 µL samples of culture. The experiment was repeated with another split culture,

where one tube was induced with 1.0 mM IPTG. The non-induced control from both of these studies was

averaged. Doubling time was calculated when cultures reached their fastest growth rate. Cells grown with

and without 0.1 mM IPTG were imaged with a Nikon phase contrast microscope (1000x).

The pSU-GhoT(4) and pSU-GhoT(7) vectors were then transformed into electrocompetent

Agrobacterium rhizogenes (15834). Transformants were verified by colony PCR for the T7-ghoT

cassette. A growth study was conducted as before on a strain carrying the pSU-GhoT(7) vector, but

cultures were grown at 25°C. As before, the culture was initially split and started at an OD600 of 0.1. One

tube was induced with 1.0 mM IPTG when the OD600 reached 1. The OD600 was measured regularly for 33

hours by taking 100 µL samples. To test cell viability, a culture was grown in the absence of inducer to an

OD600 of 1 for dilution plating on 0.1mM IPTG plates. Based on the work of Myers et al.78, this

approximately represents a cell density of 0.9 x 109 cfu mL-1 for A. rhizogenes. Dilutions were plated in

triplicate on both 0.1 mM and control plates at expected colony yields of 100 and 1000 colonies per plate.

At the end of the growth inhibition studies, a sample of induced cells was preserved for western blot

analysis. Whole cell lysate was run on an SDS gel and blotted on nitrocellulose paper. Anti-his tag

antibody staining was used to visualize the desired protein band to verify protein expression.

Finally, the pSU-GhoT(7) vector was transformed into electrocompetent A. tumefaciens cys-32

(EHA105) from the Curtis lab collection. Transformants were verified by colony PCR as before. A split

29

culture growth study was repeated, as with A. rhizogenes, and 1.0 mM IPTG induction occurred at an

OD600 of 0.1. Cells were grown at 25°C and measurements were taken regularly for 24 hours.

Assembly of pSU-empty

After the previous studies, it was desirable to construct a similar vector to pSU-GhoT with a MCS

in place of the ghoT gene so that additional toxins could be tested. To accomplish this, the plasmid was

reconstructed by Gibson Assembly in an identical manner as before, but primer A20c was used to amplify

the lacI-T7RNAP cassette. This was meant to correct the mutations observed previously. After

transformation of chemically competent E. coli TOP10, ten transformants were screened by colony PCR.

Of these, one was positive for the T7-ghoT cassette.

Due to the location of unique restriction enzymes on pSU-GhoT (noted in bold in Figure 11), the

T7 terminator had to be removed along with ghoT when adding a MCS. To build the new MCS, two

complementary oligonucleotides T18 & T19 were designed that provide PacI, EcoRI and SpeI restriction

sites when annealed; NcoI and BamHI sticky ends flanked the borders. Once annealed, the MCS insert

was ligated into pSU-GhoT, digested with NcoI and BamHI. The resulting intermediate was named pSU-

MCS (see Appendix B). This plasmid was verified by colony PCR, using primer pair T18 & A35. Next,

the T7 terminator was restored by amplifying if from pET28b-GhoT with primer pair T1 & T2, adding

PstI and HindIII sites to the ends. The PCR product was subsequently digested with these enzymes and

ligated into pSU-MCS, digested with the same enzymes. The ligation product pSU-empty (see Appendix

B and Appendix C for plasmid map and cloning strategy, respectively) was transformed into E. coli

TOP10 and verified by colony PCR with primer pair T1 & A35. Subsequently, a 50 mL culture of E. coli

harboring pSU-empty was harvested for plasmid production. Following alkaline lysis, plasmids were

isolated by standard phenol/chloroform extraction methods to yield approximately 1 mg of DNA. After

attempts to express enhanced green fluorescent protein (EGFP) with this vector were unsuccessful, it was

30

subjected to extensive sequencing to verify the issue. Although the T7 expression cassette (Figure 12)

had assembled as intended, a 3.5 kilobase deletion within the lacI-T7RNAP was detected. This truncated

plasmid was designated pSU-trunc (Appendix B) and scrapped for parts while building later plasmids.

Figure 12. Empty T7 expression cassette designed for versatile gene insertion. Unique RE sites are

shown in bold.

Assembly of pTara1300

To tighten and reduce the basal expression of T7RNAP, a different approach was adopted. A new

vector was conceptualized, on which T7RNAP would be operatively linked to the PBAD regulatory

sequence as described previously with pTara50. In order to use the T7 expression cassette originally

designed for pSU-empty, several restriction enzyme sites needed to be removed from the araC-T7RNAP

cassette. This was easier done using pTara500 as a starting point because it lacks a problematic EcoRI site

between PBAD and T7RNAP. The pTara500 vector (Appendix B) was a gift from Matthew Bennett

(Addgene plasmid #60717)51. From this plasmid T7RNAP was amplified with primer pair T39 & T40,

adding SacI and HindIII flanking sites and restoring the native T7RNAP ribosomal binding site. This

PCR product was then ligated back into pTara500, both digested with SacI and HindIII. The ligation

product was transformed into electro-competent E. coli TOP10, and four transformants were screened by

colony PCR with primer pair A37 & T44. The resulting vector was named pTara1300 (Figure 13).

Because difference between pTara1300 and its pTara500 was very minimal, the plasmid was sequence

verified at the edges of the T7RNAP gene.

31

Figure 13. The pTara1300 vector was created from pTara500 to eliminate unwanted RE sites.

Minimizing the pVS1 Replicon

To avoid using the entire pCAMBIA1300 backbone as was done in building pSU-GhoT, a smaller viable

backbone was designed. This plasmid is intended for use as a binary vector; in fact, it harbors a 2.7

kilobase T-DNA region for transfecting desired genes into plants. As this is not relevant to the

construction of a conditionally lethal vector, it could be eliminated. Additionally, there is another 1,299

base pairs between the T-DNA right border and essential pVS1 StaA gene. Less significant, there is 424

base pairs between the T-DNA left border and essential kanamycin resistance (KanR) gene. Because the

StaA and KanR open reading frames (ORFs) point away from the T-DNA, their promoters are somewhere

within these putatively unessential regions. Prokaryote promoter prediction software (available at

Fruitfly.org) was used to determine the most likely location of these two promoters79. From these results,

a 3.9 kilobase region was deemed nonessential (see Figure 14); the predicted promoters were buffered

with roughly 150 to 200 base pairs of DNA in case there might be unknown transcription factor binding

sites. Interestingly, a cryptic ORF for a 25 kDa protein was found upstream of the StaA gene, which

32

contained domains consistent with members of the recombinase (resolvase) protein family. According to

Heeb et al., this gene is unnecessary for stable maintenance and replication of the pVS1 replicon26. In a

later experiment, the minimal backbone was tested for stable transformation in A. tumefaciens EHA105.

Figure 14. The nonessential region (gray) was removed in subsequent versions of the suicide vector.

Assembly of pSU16R and pSU16F

With the minimal backbone determined and araC-T7RNAP cassette perfected, these two

elements could be consolidated into a single vector along with an empty T7 expression cassette. The

pCAMBIA1300 backbone was amplified with primer pair T48 & T28. This PCR product was

subsequently digested with DpnI to eliminate any pCAMBIA1300 template. The araC-T7RNAP cassette

was amplified from pTara1300 with primer pair T46 & T47. Lastly, the empty T7 expression cassette was

amplified from pSU-trunc. The primers used in the three preceding reactions added overhangs to each of

the fragments for Gibson Assembly; also, an XhoI site was added between the T7RNAP and PT7. The

three fragments were purified directly through PCR purification and joined using NEBuilder® HiFi DNA

33

Assembly master mix (NEB). The femtomole ratio used was 32:32:96 (pCAMBIA1300 backbone:araC-

T7:T7 expression cassette). After a 1 hour assembly at 50°C, the reaction was immediately transformed

into electro-competent E. coli TOP10. Some colonies tested positive when screened with primer pair T42

& T13. A second and third round of colony PCR with primer pairs A37 & T14 and T7F & T14 were not

conclusively positive for any colonies. The desired plasmid product from this assembly was pSU16 (refer

to Appendix B and Appendix C for plasmid map and cloning strategy, respectively). One colony was

selected for sequencing, which revealed a 4.2 kilobase deletion within the araC-T7RNAP cassette. This

truncated vector was named pSU13-trunc (Figure 14). Fortunately, one MfeI site from the araC-

T7RNAP cassette and the added XhoI site were preserved. The pSU13-trunc vector alongside

pCAMBIA1300 was transformed into A. tumefaciens EHA105 to determine whether the backbone was

sufficient for broad host range replication. A comparable number of transformants of each vector was

obtained under kanamycin selection, demonstrating that the backbone was sufficient.

Figure 15. Map of pSU13-trunc. Note the MfeI and XhoI restriction sites for correction of the araC-

T7RNAP deletion

34

After it became apparent that the orientation of the T7RNAP gene relative to the T7 expression

cassette may be an issue for cloning this vector, the T7 expression cassette was flipped. The cassette was

amplified from pSU13-trunc using primer pair T55 & T56, reversing the flanking restriction sites for

XhoI and BspEI. This was ligated back into pSU13-trunc, which was digested with the same enzymes.

This was transformed into electro-competent E. coli TOP10, and transformants were verified by colony

PCR using primer pair T14 & T54. The resulting plasmid was named pSU13R (Appendix B), and

sequence verified.

To restore the araC-T7RNAP cassette, the construct was inserted into both pSU13R and pSU13-

trunc in parallel. The araC-T7RNAP cassette was amplified from pTara1300 with primer pair T47 &

T57, and then digested with EcoRI and XhoI. Both pSU13R and pSU13-trunc were simultaneously

digested with MfeI and XhoI, followed by a treatment with Antarctic Phosphatase (NEB). Using a 23:46

femtomole ratio of vector to insert, the fragments were ligated for 8 hours at 16°C. The two products were

transformed into chemically competent E. coli TOP10, giving twice as many colonies for the plasmid

with the reversed T7 expression cassette. The reversed expression vector was named pSU16R (Figure

16), while its counterpart was named pSU16F (Figure 17). Both constructs were verified using primer

pair T42 & T13. A second round of colony PCR using primer pair A37 & T7F verified pSU16R.

Similarly, primer pair A37 & T54 verified pSU16F. Colonies were consistently positive or negative in

both rounds of screening. Positive colonies of each vector were re-streaked and subjected to a final PCR

confirmation with primer pair T45 & T43. Colonies 1 and 2 of pSU16F, and colonies 2 and 4 of pSU16R

were cryopreserved and have yet to be sequenced.

35

Figure 16. Map of pSU16R. Orientation of PT7 is flipped relative to pSU16F.

Figure 17. Map of pSU16F.

36

Preparation of Toxins for Insertion

While constructing the empty vectors for toxin expression, toxin genes were prepared for

insertion based on the conserved MCS of the desired suicide vectors. Barnase was isolated from

pMT1002 (see Figure 7), a gift from Robert Hartley (Addgene plasmid # 8621)36. The gene was PCR

amplified using primer pair T7 & T8, and directly purified. The construct was digested with NcoI and

EcoRI, ready for insertion into the inducible vector.

Similarly, Tse1 was isolated from pET22b-Tse1 (see Figure 8), a gift from Joseph Mougous67.

Because the toxin gene contains a natural NcoI site within its sequence, it was amplified with primer pair

T11 & T12 for Gibson Assembly. The PCR product is intended for insertion into a vector, linearized at

the NcoI site.

37

Chapter 4 :

Results and Discussion

Selection Pressure against Lethal Constructs

In generating the pSU-GhoT vector, mutations were observed in all clones. Even under catabolite

repression with glucose, it is reasonable to assume that these mutations conferred some sort of relief to the

cells by helping to escape the lethal effects of basal toxin expression. The mutations in pSU-GhoT(4) and

pSU-GhoT(7) are shown in Figure 18. For pSU-GhoT(4), there was a single point deletion in PT7, yet the

ghoT open reading frame was left fully intact. This mutation was anticipated to be more deleterious to the

expression system than that of pSU-GhoT(7). T7RNAP is highly specific to its respective recruitment site

at PT7; the system evolved within bacteriophage T7 to organize an aggressive and rapid assault on its host.

Also, identical PT7 consensus sequences occur throughout the genome of this phage. Chapman et al.

showed that single mutations within the promoter sequence can reduce expression to less than 10% of WT

levels80. The deletion in pSU-GhoT(4) shifts nine of the nineteen nucleotides a space to the right; several

of these changes alone have been shown to abolish function. In pSU-GhoT(7), a single nucleotide shift

the T7RNAP ORF toward the end of the gene. This changes the last four native amino acids of the gene

and extends the ORF for an extra twelve amino acids overall. From the crystal structure, the C-terminus

of T7RNAP appears to be far from the active site, but this mutation might still affect the integrity of the

hydrophobic core of the protein81. Additionally, there is a mutation in the ghoT gene that changes the

second amino acid from an alanine to a glutamate residue. This amino acid was not determined to be part

of an active site, unlike Phe3860. Although GhoT is a membrane protein, the N-terminus was predicted to

be cytoplasmic-oriented rather than part of either of the two transmembrane domains predicted using the

Phobius transmembrane topology predictor (Appendix G). For these reasons, the mutations of pSU-

GhoT(7) were presumed to be less deleterious to the function of the construct, and this vector was used in

subsequent experiments.

38

Figure 18. Alignment of mutations in pSU-GhoT(4) and pSU-GhoT(7) against expected sequence (top).

Although these mutations may be attributed in part to the incorrect sequence of primer A20, it is

likely that this error acted more as an escape route for some cells to survive selection. Therefore, when

this primer was corrected, the chance of incorrect assembly decreased. In turn, less colonies were able to

escape selection during the second attempt to assemble pSU-GhoT, and the number of positive

transformants dropped to one. Moreover, the only positive clone was later determined to have a ruinous

deletion of the lacI-T7RNAP cassette. Exploiting these events, one of the clever uses for lethal vectors is

for the entrapment of insertion sequence (IS) elements. These mobile sequences carry only the genes

necessary for transposition throughout the genome; and one mechanism by which a cell might survive

positive selection is if one of these elements inserts itself into the lethal marker72. Similarly, the

occurrence of mutations despite the use of a high-fidelity polymerase would suggest that the basal

expression of T7RNAP, and in turn, the GhoT toxin under the control of the lacUV5 promoter is

sufficiently high to create a significant selection pressure. In fact, the lacUV5 promoter gives higher

expression levels than the native Plac due to the inactivation of the CAP binding site. Because the presence

or absence of CAP no longer affects recruitment of RNAP, regulation of expression is rendered

insensitive to catabolite repression. Methods developed to counter this type of lax expression will be

discussed in the following section.

When E. coli harbored pSU-GhoT(7), 0.1 mM IPTG was not sufficient to completely halt cell

growth. As shown in Figure 19, this concentration resulted in slower growth and a lower density overall;

however, the OD600 continued to increase with a doubling time of 3.0 hours, less than that of the control at

1.6 hours. On the other hand, cells induced with 1.0 mM IPTG were not able to grow above an OD600.

This trend is consistent with experiments by Wang et al., testing IPTG-induced expression of ghoT in E.

coli59.

4 7

39

Figure 19. GhoT expression using pSU-GhoT(7) in E. coli. Induction occurred at time zero.

Visual comparison of the cultures clearly showed a difference in the density of cells, where the

induced culture was much more translucent (Figure 20). Microscopy revealed that induced cells were

polarized in morphology; many with the transparent ghost phenotype were more elongated, while there

was also a larger number of compact spherical cells (Figure 21).

Figure 20. Culture comparison of induced (left) versus un-induced (right).

0

0.5

1

1.5

2

0 1 2 3 4 5 6

OD

60

0

Time (hr)

Control

0.1 mM IPTG

1 mM IPTG

40

Figure 21. Cell morphology differences between induced (left) versus un-induced (right) at 1000x

magnification.

Although it is established that overexpression of GhoT destabilizes the membrane of E. coli60, it

was of great interest to see if this effect was also true for Agrobacterium. When A. rhizogenes harboring

pSU-GhoT(7) was induced with 1.0 mM IPTG, the doubling time of the culture was roughly half that of

non-induced cells (Figure 22). Because this difference in turbidity was not conclusively a result of cell

death, or perhaps instead just the metabolic load on the cells from overexpression of a protein, additional

studies were performed. Dilution plating of a culture on IPTG plates revealed little difference in viability

between induced and non-induced cells (Figure 23). To confirm, GhoT production in the A. rhizogenes

that survived induction, whole cell lysate was used in western blot analysis. The western blot (Figure 24)

revealed a band, consistent with the size of GhoT+6xhis (7.6 kDa). These results verified that the protein

was most likely being expressed in A. rhizogenes following IPTG induction. Combined with the viability

tests, this suggests that GhoT is not particularly toxic to A. rhizogenes even at high levels in the cell.

Instead, this bacteria may be a suitable expression platform for the expression of GhoT, which has yet to

be expressed at high levels in E. coli without causing cell death. This is of particular importance to those

currently attempting to characterize the protein.

41

Figure 22. GhoT expression using pSU-GhoT(7) in A. rhizogenes. Induction time is denoted by ▼.

Figure 23. Dilution plating shows a slight decrease in viability when expression of GhoT is

induced.

0

0.5

1

1.5

2

2.5

0 5 10 15 20 25 30 35

OD

60

0

Time (hr)

Control

1 mM IPTG

0

50

100

150

200

250

~100cfu/ml ~1000cfu/ml

Co

lon

ies

Control

+IPTG

42

Figure 24. Anti-his tag immunoblotting of induced A. rhizogenes whole cell lysate. Although a negative

control was not run, this is the expected size of GhoT+6xhis.

Interestingly, the growth of A. tumefaciens carrying pSU-GhoT(7) was not adversely affected by

IPTG induction (Figure 25). Both the induced and non-induced cultures had a doubling time of 2.5 hours.

Figure 25. GhoT expression using pSU-GhoT(7) in A. tumefaciens. No difference in growth.

The GhoT toxin did not elicit the same response in both Agrobacterium spp. as was seen in E.

coli. The protein is normally secreted and incorporated into the membrane of the latter, while the former

bacterial species may not have secretion machinery that recognize this foreign protein. With insertion into

the membrane disrupted, it would then build up within the cytoplasm where it is unable to exert its lethal

effects. Additionally, GhoT is not thought to simply be a structural protein, but may interact with several

10-

15-

GhoT7.8 kDa

0

0.5

1

1.5

0 10 20

OD

60

0

Time (hr)

Control

1 mM IPTG

43

potential targets. A mutation of phenylalanine 38 to arginine yields a non-toxic GhoT mutant, yet it still

localizes within the membrane like its WT counterpart; therefore, this residue is thought to play a role in

interacting with other protein targets60. If this is the case, then the effects of GhoT may be quite host-

specific, depending on its interactions with additional proteins in the cell. Lastly, the in vivo function of

GhoT is not necessarily to be a potent toxin, but rather a mediator of cell stress and player in

bacteriostasis. In contrast, the effector proteins Tse1 and barnase were specifically selected for eventual

testing because of their roles as powerful bactericides.

Tightening Expression Control

The lacUV5 promoter was insufficient in repressing basal expression of the GhoT toxin until

induction was desired. For this reason, the more stringent araC-PBAD regulatory sequence was employed

in this study. Alternatively, some strains of BL21 have been developed that carry a vector, compatible

with pET vectors, encoding a fragment of the T7 lysozyme gene. This protein product is an inhibitor of

T7RNAP and can be used to decrease levels of basal expression by minimizing the activity of T7RNAP

until induction82. In fact, these strains have been shown to facilitate the cloning of toxic gene products that

proved unsuccessful otherwise37. This was the method employed when testing pET22b-Tse167. Although

the lacUV5 form is poorly suited for tight gene regulation, modified versions of Plac exist that provide

much greater control. As mentioned, this inducible system is regulated by the repressor protein lacI. The

lacIQ allele is a mutant form of the gene, where its constitutive expression is increased 10-fold due to a

promoter mutation39. A symmetrical lacO can also be used to increase binding of the repressor, further

restricting access to the RNAP recruitment site83. The review by Saïda et al. describes a variety of useful

genetic tools that have been engineered for the purpose of expressing difficult proteins.

In addition to genetic elements, culture conditions can be used to reduce toxic gene expression. E.

coli grow optimally at 37°C, but suboptimal temperatures (15-30°C) can be used to reduce bacterial

44

metabolism and protein production. At these temperatures, it can be easier to maintain the stability of

lethal elements. Similarly, suboptimal culture conditions on minimal media with an energy-poor carbon

source like glycerol increases bacteriostasis, as a stress response. In this sluggish state, more resources are

needed for de novo synthesis of primary metabolites, meaning less resources are available for the

expression of non-essential genes50. For E. coli, 0.3% glucose can be added to LB media to increase

catabolite repression. In the case of the araC-PBAD regulatory mechanism, D-fucose acts as a competitive

inhibitor of arabinose to modulate expression of the regulon84. At scale, the use of this somewhat rare

sugar would be cost-prohibitive. As observed when this regulatory element was used in A. tumefaciens,

the inhibitory effects of D-fucose were almost equal to inhibition by Citric Acid Cycle intermediates,

primarily malate, fumarate and succinate46. Unlike D-fucose, malate would not be a major limitation to

growing large-scale cultures of A. tumefaciens with a conditional kill switch.

Vector Versatility

For building a versatile vector for inducible expression, the plasmid backbone was minimized for

ease of DNA manipulations. Consistent with previous studies, the minimal backbone designed was

sufficient for stability and replication in A. tumefaciens. Although the cryptic resolvase ORF is

unnecessary, a small ORF that codes for a 7.7 kDA protein (designated ParG) following StaA is necessary

to maintain stability26. Transformants also survived kanamycin selection without spontaneous mutations

around the KanR gene, showing that the buffer region was sufficient. The pSU13-trunc, pSU13R,

pSU16F and pSU16R vectors harbor the necessary elements for conjugation or transformation into a wide

range of hosts. Although the two pSU13 series vectors lack the araC-T7RNAP cassette, they harbor a T7

expression cassette with an empty MCS. These would be quite useful as broad host range vectors or

binary vectors for T7-directed expression; T7RNAP would need to come from either the chromosome or

45

another vector. These vectors would function well alongside the aforementioned T7RNAP transposon

system52.

The pSU16R was created as an improved version of pSU16F. As mentioned, several issues arose

when attempting to clone pSU16F, including large truncations and a lack of transformants. Because this

vector does not contain a gene within the T7 expression cassette, it was not expected to be harmful to

bacteria. A possible reason for these cloning failures was linked to the orientation of T7RNAP with

respect to the T7 expression cassette. It was assumed that the terminator from bacteriophage T7 is

efficient in halting transcription of T7RNAP; however, this is not the case as the termination efficiency of

this element is only 74%85. Within the genome of the phage, genes 11 and 12 do not have a promoter

between the start of the gene 11 ORF and the end of the T7 terminator; therefore, both of these essential

genes depend on T7RNAP read-through to be transcribed. When a T7 expression cassette was integrated

into the genome of E. coli, T7RNAP-directected expression led to an upregulation of the genes within 32

kb on the sense strand86. With plasmid-based systems, this could pose a major problem in the case that

T7RNAP transcribes antisense mRNA to essential genes on the plasmid backbone. Additionally, the

generation of large amounts of off-target transcripts increases the metabolic load on the cell without

increasing production of desired protein. Because pSU16F harbors both T7RNAP and a T7 expression

cassette in the same orientation, it means that read through would cause T7RNAP to transcribe its own

gene. Even one protein that manages to circle around the entire plasmid could induce an autocatalytic

cycle that would surely halt cell growth. By shrinking the size of the plasmid to under 10 kb, this abberent

result is even less of a challenge. Therefore, the orientation of the expression cassette was flipped, so that

even minimal amounts of basal T7RNAP read through would not set off such a chain reaction.

Interestingly, this issue is quite similar to the problems experienced when first cloning the T7RNAP gene

from the T7 phage genome due to a PT7 downstream48.

Although experiments have yet to be conducted, cultures with pSU16F grow noticeably slower

and are less turbid compared to cultures with pSU16R. This has made it difficult to prepare this plasmid

46

for sequence verification. If indeed this is an issue, Mairhofer et al. has developed terminator

combinations that have up to a 99% termination efficiency87. On the other hand, pSU16R should not

exhibit these problems to the same degree because the cassettes are in opposite orientations. This vector is

valuable as a broad host range expression vector with a self-contained T7 expression system. The MCS of

pSU16R is given in Figure 26. For working towards an improved inducible kill switch, this vector is

designed so that almost all of its elements can be switched out and replaced. It is the ideal system for

testing high-level protein expression in a wide range of organisms, as well as verifying regulator

stringency.

Figure 26. Multiple cloning site of pSU16R. After inserting, a gene of interest after PT7, this vector could

be used to express the target protein at high levels in a variety of hosts.

47

Chapter 5 :

Future Work

Naturally, the first step in continuing this work would be to sequence verify the pSU16R and

pSU16F expression vectors. Growth inhibition experiments would then be used to determine whether

induction of the empty vectors creates a noticeable load on the cell. As mentioned, E. coli harboring

pSU16F has been qualitatively observed to be much less turbid when grown side by side with its

counterpart. Controlled experiments would characterize this phenomenon.

Regardless, the intended purpose of these vectors was to create an inducible kill switch to be used

to eliminate cultures of Agrobacterium harboring the lethal construct. In this regard, the barnase and Tse1

toxins mentioned above would be cloned into pSU16R to demonstrate induction of host cell death. The

toxins that have been amplified will be inserted to generate pSU16R-Barnase (Figure 27) and pSU16R-

Tse1 (Figure 28).

Figure 27. Map of pSU16R-Barnase

48

Figure 28. Map of pSU16R-Tse1

Though unlikely, it may be the case that the PelB signal is not effectively recognized by

Agrobacterium in directing Tse1 to the periplasmic space. In this event, alternate secretion peptides that

work in Agrobacterium will need to be characterized. The chvE and virJ genes, native to Agrobacterium

are known to be localized in the periplasmic space88,89. Currently, fusion of the predicted ChvE signal

peptide to Tse1 by overlap extension PCR is being attempted to avoid this type of issue. Finding a host-

specific toxin that is ineffective in E. coli, yet lethal in Agrobacterium, would greatly increase cloning

ease. Despite the measures taken to reduce basal expression, it is likely that this will continue to be a

challenge when selecting transformants and culturing strains that harbor a suicide vector. To mitigate this

issues, minimal medias like M9T (see Appendix F), supplemented with an expression inhibitor as the

carbon source would be recommended. If transformation failures and frequent mutations continue to be

problem, the system will need to be modified. Deliberately, the pSU16R vector is designed so that most

of its elements are interchangeable due to the addition of unique restriction sites between them. Currently,

incorporation of an enhanced lacI cassette is being attempted, which uses the lacIQ promoter and

49

symmetrical lacO. Alternatively, the specific immunity or antitoxin protein could first be added to the

plasmid under the control of a weak promoter before the toxin is inserted; this relief would then be easily

overpowered when T7 expression is fully induced. Note that if an antitoxin is added, it should be

expressed in the antisense direction in relation to the T7 expression cassette; otherwise, induction of the

toxin may also increase expression levels of the antitoxin. Once the system is perfected, it is ultimately

meant to be integrated into the genome of the host species.

For integration, methods such as those used by Hood et al. in generating deletions in the

Agrobacterium Ti plasmid would be employed12. A selectable marker would first need to be added into

the construct intended for genomic insertion. The construct would then need to be flanked with regions

homologous to the desired site of insertion. To force integration, the plasmid would need to be

nonreplicable in Agrobacterium (via removal of the pVS1 replicon elements). Alternatively, the construct

would be moved to a broad host range plasmid with a well-characterized incompatibility group, unlike

pVS1. For example, a vector with an RK2-derived ori (IncP1 incompatibility group) would be used to

move the kill switch into Agrobacterium25. A second, incompatible vector would then be introduced with

a different selection to evict the first vector for the purpose of identifying successful cross over events.

Once stable integration is achieved, the Agrobacterium would be tested at scale in plant cell bioreactors

for transient protein expression. Induction of the kill switch would be tried and tested to determine the

viability of this method when working with an inherently larger number of potential mutant bacteria cells

that may escape counterselection.

In any case, the pSU16R vector constructed herein is of great significance for its potential use as

a broad host range, high-expressing system. It is a self-contained system, carrying all of the elements

necessary for T7-directed expression on a relatively small vector. The presence of the bom and pVS1 ori

allows the vector to be replicated in a variety of gram-negative hosts. Future studies would involve testing

this system in several diverse bacteria, a task for which the Curtis lab group is well-equipped.

Additionally, any system capable of producing high levels of a toxic protein is valuable in itself for the

50

purpose of protein purification, X-ray crystallography and characterization. Overall, the vector is

deliberately versatile.

51

Appendix A

List of Primers

Table 1. Relevant Primer and Oligonucleotide Sequences

Abbr. Sequence*

A17 TATGACCATGATTACGAATTTCCCGGACACCATCGAATGG

A20 TATAGTGAGTCGTATTAATTTTACGCGAACGCGAAGTCCG

A20c TATAGTGAGTCGTATTAATTTACGCGAACGCGAAGTCCG

A21 CGGACTTCGCGTTCGCGTAAAATTAATACGACTCACTATAGGGGA

A22 CCGGGTACCGAGCTCGAATTATCCGGATATAGTTCCTCCTTTC

A30 TCCCGGACACCATCGAATG

A33 TTACGCGAACGCGAAGTCC

A35 AACCCTGTGGTTGGCATGCA

A37 CTGATTCACGACTCCTTCGGTACCA

M13F GTAAAACGACGGCCAGT

M13R CAGGAAACAGCTATGAC

T1 ATAGCTGCAGATCCGGCTGCTAACAAAGC

T2 ACAGAAAGCTTCCCATTCGCCAATCCGG

T7 GCTTCCATGGGCGCACAGGTTATCAACACGTTT

T7F TAATACGACTCACTATAGGG

T7R GCTAGTTATTGCTCAGCGG

T8 CGTAGAATTCTTATCTGATTTTTGTAAAGGTCTGATAATGGTC

T13 CGTTCCACATCATAGGTGGTCC

T14 GACAACGGTTAGCGGTTGA

T18 CATGGTTAATTAAGAATTCACTAGTG

T19 GATCCACTAGTGAATTCTTAATTAAC

T28 CCAGGACGAACCGTTTTTCATTACC

T33 GGTAATGAAAAACGGTTCGTCCTGGCCCATTCGCCAATCCGG

T39 TGCTAAGCTTTTACGCGAACGCGAAGTCCGAC

52

T40 CGCTGAGCTCAAGAGGATACCATATGAACACGATTAACATCGCTAAGAAC

T42 TGAATGATGTAGCCGTCAAGTT

T43 GCTATGCCATAGCATTTTTATCC

T44 TCAGGCTGAAAATCTTCTCTCA

T45 TAATGGTGATGTACGCTACGG

T46 AATCAACATGCTAATGTGCCTGTCAAATGGACGAAGCAG

T47 TGAGTCGTATTAACTCGAGAAAAATAAACAAAAGAGTTTGTAGAAACGC

T48 TGACAGGCACATTAGCATGTTGATTGTAACGATGACAGAGC

T49 TTTTGTTTATTTTTCTCGAGTTAATACGACTCACTATAGGGGAATTGTGA

T54 TTTCTAGAGGGGAATTGTTATCCG

T55 CGATGTCCGGATTAATACGACTCACTATAGGGGAATTGTG

T56 TCTTGCTCGAGTTCCTCCTTTCAGCAAAAAAC

T57 TAGCTGAATTCCGTCAAGCCGTCAATTGTCTG

*All primers were purchased through IDT or PSU dnaLIMS

53

Appendix B

Additional Plasmid Maps

Figure 29. Map of pSU-MCS

Figure 30. Map of pSU-empty.

54

Figure 31. Map of pSU-trunc

Figure 32. Map of pTara500.

55

Figure 33. Map of pSU16

Figure 34. Map of pSU13R.

56

Appendix C

Cloning Strategy Maps

pCR4-lacI-T7RNAP

pSU-GhoT

pSU-empty

pTara1300

pSU16

pSU13R

pSU16R

pSU16F

Appendix D

Gibson Assembly Details

pSU-GhoT

Fragment 1: lacI-T7RNAP amplified with primers A17 & A20

Fragment 2: T7-ghoT amplified with primers A21 & A22

Vector: pCAMBIA1300 linearized at EcoRI

pSU16

Fragment 1: araC-TRNAP amplified with primers T46 & T47

Fragment 2: T7-exp amplified with primers T49 & T33

Vector: pCAMBIA1300 amplified with primers T28 & T48

pSU16R

Fragment: Tse1 amplified with primers T11 & T12

Vector: pSU16R linearized at NcoI

Appendix E

Gibson Assembly: Design and Execution

Overview

The traditional genetic engineering technique of restriction enzyme cloning (cut/paste)

comes with many limitations, primarily the need for unique restriction sites within the sequences

of your DNA fragments. Essentially, the Gibson assembly is a far superior method for achieving

the same results with fewer drawbacks and several benefits:

Less reagents required = lower cost

Less time required

Able to join up to 8 fragments in a single reaction

No DNA scars flanking the insertion site

High efficiency for achieving desired construct

Although each cloning scheme is unique, depending heavily on the DNA fragments involved, the

Gibson assembly is highly dynamic to accommodate for these variations. I will navigate through

both the wet lab techniques and in silico DNA modeling necessary to engineer your way to

success. Hopefully, my standardized instructions can help you through the process of assembly

to create your novel vector or construct. This standard operating procedure will cover the

following topics:

Polymerase Chain Reaction (DNA fragments are created)

Gibson Assembly reaction (fragments are combined)

Transfection (the synthesized vector is directly inserted into cells)

Reagents, Templates & Tools

Note: It is assumed that most of these materials are available & prepared beforehand as is typical

in most molecular biology labs

An idea of what DNA fragments you want to join!

SnapGene (or similar DNA editing software like Benchling)

NanoDrop spectrophotometer

Thermocycler

1000 µl, 200 µl, 20 µl, 10 µl Micropipetters with tips

0.2 ml PCR tubes

1.5 ml Eppendorf tubes

Tube racks

Reagents for PCR:

o Phusion DNA polymerase and Phusion Buffer from NEB

o Template DNA containing your genes, plasmid, or sequences of interest (DNA

can be acquired from AddGene, synthesized by GeneWiz, etc.)

o Primers can be synthesized and purchased on campus through Penn State

University LIMS or Integrated DNA Technology

o Nucleotides (mixed dNTPs)

o Sterile Water

Reagents for Gibson Assembly (purchase or refer to recipe in Appendix E):

o Gibson Assembly Master Mix from NEB (contains all necessary enzymes and

buffers)

o Sterile Water

o Amplified fragments from PCR

Petri dishes of LB medium with antibiotics & SOC liquid

Sterile environment with chemically competent bacteria cells (Typically, E. coli)

Incubator at 37ºC

Estimated Cost & Time Needed

Most of these tools and supplies are standard for any molecular biology lab. Free DNA

editing software is available through the links above. You will need to purchase the templates if

they are not readily available, which typically costs around $65-100 or more when utilizing a

gene synthesizer. Primers are priced per base pair and typically cost between $6-12 per sequence.

Two primers are needed for every DNA fragment you want to join. Gibson assembly reagents

are the main ingredients that must be purchased, or can be home-made according to the recipe in

the following chapter. Fortunately, New England Biolabs supplies a mix of all the necessary

enzymes and buffers, which is the definitely the easiest option. Remember that a single reaction

is able to join multiple pieces of DNA; still, the master mix will be the most expensive

ingredient.

In total, the procedure will cost the laboratory between $100 – 250 per reaction, starting

from scratch. More fragments will mean more templates and primers. Compared to restriction

enzyme cloning that would also require the same amount of primers and templates, this method

costs considerably less!

For time, the use of computer programs will take about 3 – 6 hours. The PCR should take

about 20 minutes to set-up and 2 – 4 hours to run. It should take around 15 minutes to measure

DNA concentration on the NanoDrop. The Gibson assembly requires 1.5 hours to set-up and run.

Finally, the bacterial transfection takes less than 3 hours. Overall, the entire procedure is best

divided over two days: one for computer planning & PCR, another for the remaining wet lab

techniques. This a huge time saver compared to using restriction enzymes, which would take 3

days per fragment!

Instructions

In Silico (For SnapGene Users)

1. Create digital files of the sequences to be viewed in your gene editing program after

selecting the fragments to be joined.

2. Import each into SnapGene. Below is an example of three fragments to be joined.

3. Go to Actions > Gibson Assembly, then select the appropriate option for your cloning

scheme. A control panel with several windows will pop-up on the screen.

4. Upload one DNA fragment file into each of the windows. They should be arranged in

the desired joining order and then oriented to point in the desired direction.

Note: For my example, I will be inserting two fragments into the third to create a

circular plasmid. I am given a panel with four windows.

5. Click the Choose Overlapping PCR Primers button in the last window. A second

panel will appear.

6. IMPORTANT: Set the target Tm for PCR primers to a value between 57 - 61ºC. For

overlapping ends, it is best to set a range of 20 to 30 base pairs with a target Tm ≥

50ºC. Clicking Choose Primers will generate the optimal primers for your Gibson

Assembly and PCR.

7. Check that your fragments are ordered and oriented correctly in the Product

viewing window before clicking Assemble. A new file will be generated to show your

Gibson Assembly construction:

8. Export the auto-generated primer

sequences and then order them from

your favorite company (PSU LIMS

will synthesize primers in about two

days).

*See Factors to Consider & Final Reminders

below for more about designing primers to

prevent any unexpected problems that may

physically interfere with the actual assembly. Certain checks can be done to make sure your

reaction is more likely to be successful, saving time in the long run.

Polymerase Chain Reaction (PCR)

1. Acquire all template/source DNA and primers. Protocol adapted from NEB.

2. Using one PCR tube for each fragment, add the following thawed reagents:

a. 10 µl Phusion Buffer (5x Stock)

b. 1.5 µl dNTPs (10 µM stock)

c. 2.5 µl of the appropriate forward primer (10 µM stock)

d. 2.5 µl of the appropriate reverse primer (10 µM stock)

e. .5 µl Phusion polymerase (2000 units/ml stock)

f. .5 - 1 µl of the appropriate template DNA (that matching primer pair)

g. Fill to 50 µl with sterile water

Notes: No DNA template is added to negative control tube, only primers!

Reagents in blue MUST be kept on ice during set-up. Add Phusion last! It is best

to prepare a mastermix of the appropriate volume to be distributed to all tubes

before adding template

3. Move tubes to thermocycler. Set the following cycle:

a. 98ºC for 1 – 5 minutes

b. 98ºC for 30 seconds

c. 57-60ºC for 30 seconds Note: Use lowest Tm of the primers used

d. 72ºC for X seconds Note: X = 15 + 0.03 × bp length of longest template

e. 72ºC for 7 – 10 minutes

Figure: Exponential amplification of DNA with PCR

30 cycles

f. ∞ Hold at 15ºC

4. Analyze 1 µl from each of the finished PCR reactions on the NanoDrop. This will

give you the concentration of dsDNA that has been amplified.

Notes: As a check, you can run 10 µl of each reaction on an agarose gel. The

negative control (no template) should give no bands under UV light. The

fragment PCR tubes should give ONLY one band corresponding to their

respective base pair length. If more than one band is observed, your primers

amplified a mixture of different DNA pieces. If so, cut out the band of the correct

size and follow standard gel purification techniques to isolate.

5. IF waiting until the next day to continue, keep PCR products in a freezer overnight.

6. Isolate fragments directly from PCR product using standard DNA purification kit.

Gibson Assembly

What’s going on at the molecular level? SnapGene generated DNA primers that added

20 -35 base pair flanks to the end of each fragment during the PCR. These flanks overlap with

the intended adjacent fragments. During the Gibson Assembly, T5 exonuclease chews away

some of the DNA to expose a single strand. This binds to its respective complementary flank on

another fragment. Polymerase replaces the missing base pairs, and Ligase glues the remaining

gaps to fully join all of the fragments. All of these enzymes are included in the NEB master mix!

Quick Calculations

Case1. One fragment in the assembly is significantly larger than the rest and called the vector.

1. Use NEBioCalculator to calculate the moles of 50-100 ng of vector.

2. Multiply this number by 2 and use as the number of moles of each fragment to use. If

a fragment is less than 500 base pairs, multiply by 5.

3. Calculate the volume of each fragment needed to maintain this ratio (1:2 and/or 1:5)

using the vector as a basis. Total amount of DNA should be 0.03-0.2 pmols.

Case2. All fragments are about the same size

1. Use the same to calculate the moles of 100 ng of the largest fragment.

2. The rest of the fragment will use this same number of moles (1:1). If a fragment is

less than 500 base pairs, multiply by 5.

3. Calculate the volume of each piece to maintain this ratio. Total amount should be 0.2-

0.5 pmols.

The Assembly

1. In a single PCR tube, add the following reagents:

a. 10 µl NEB Gibson Master Mix (refer to appendix F for home-made recipe)

b. Add the volume of each fragment as calculated above

c. Fill to 20 µl sterile water

Note: Total volume of fragments in (b) should be less than or equal to 4 µl

2. Put tubes in thermocycler. Set machine at 50ºC for 1 hour followed by 15ºC hold.

Transfection

Note: This procedure is ONLY suitable when the total size of your assembled plasmid with all

fragments joined is less than 20,000 base pairs. If larger, more extreme measures like

electroporation are required to coerce the bacteria into taking up your DNA.

1. Take 4-7 µl of the finished Gibson reaction. Add this to the chemically competent

bacteria in an Eppendorf tube. Let sit on ice for 30 minutes.

2. Quickly put tube in 42ºC for 45 seconds. Then move directly onto ice for 2 minutes.

3. Add 500 µl growth media SOC to the tube. Let sit for 1 hour in 37 ºC incubator.

Note: This step allows the weakened bacteria to build up strength and begin

expressing antibiotic resistance genes conferred by your plasmid!

4. Plate 200 µl onto a petri dish with the appropriate antibiotic media. Place dish in

37ºC incubator overnight. If the procedure was successful, colonies will begin to

grow by the next day!

Factors to Consider & Final Reminders

Primer design may seem quick and easy with SnapGene, but it is not always perfect!

When the flanking ends of your fragments contain a high density of G and C base pairs (>60%

G/C content), the annealing temperature of the overlaps can be affected. This means that the

program will make an overlap of less than 20 base pairs, but still have the target Tm of 50ºC.

Without enough base pair overlap, the efficiency of the Gibson Assembly greatly decreases!

Also, if the DNA contains any palindromic sequences or repeats, it

may form obstructive secondary structure when the T5 exonuclease

chews the ends. Online DNA secondary structure prediction

software can be used to avoid this issue before it can cause any

problems. For example, a flank with the sequence

CGCAAAAAAAAAGTACTTTTTTTTTT becomes:

25+ base pair overlap!

If your PCR fails to yield any DNA, this is most likely caused by a primer error. In most

cases, this is because the template sequence is incorrect—I rarely receive DNA that is

completely identical to the given sequence. This is the most frustrating part about working with

DNA! Always sequence your templates around the regions of interest for verification. Positive

controls can be used to diagnose an actual failure of the PCR caused by missing reagent,

thermocycler malfunction or enzyme degradation.

Still, both PCRs and Gibson Assembly are highly efficient and rarely fail when all of the

ingredients are present (even if the concentrations are significantly off). Once your colonies

grow, harvest some of the plasmids and send them for Sanger sequencing of the overlap

regions. If there are any errors or mutations caused by the assembly, they will usually be in the

overlaps! Another issue that I have encountered is transfection failure due to genes in the

assembled plasmid that are toxic to bacteria.

Overall, the Gibson Assembly is a highly effective technique for rapid recombination of

DNA fragments. These instructions are meant to help your cloning to go as smoothly as possible

while saving some time and money!

Appendix F

Media and Reagent Recipes

Gibson Assembly Master Mix

This recipe was developed by Gibson et al. to demonstrate the assembly of DNA up to several hundred

kilobases90. The master mix is meant to be used in a ratio of 3:1 (V:V) with DNA; for example, 5 µL of

DNA would be added to 15 µL of Assembly Master Mix.

5x Isothermal Reaction Buffer (6 ml)

1 M Tris-Hcl (pH 7.5) 3 ml

1 M MgCl2 300 µL

100 mM dGTP 60 µL

100 mM dATP 60 µL

100 mM dTTP 60 µL

100 mM dCTP 60 µL

1 M DTT 300 µL

PEG-8000 1.5 g

100 mM NAD 300 µL

ddH2O fill to 6 ml

Store at -20°C

Assembly Master Mix (1.2 ml)

5x Isothermal Buffer 320 µL

40 U/µL Taq ligase 0.16 µL

1 U/µL T5 exonuclease 0.64 µL

2 U/µL Phusion Pol 20 µL

ddH20 fill to 1.2 ml

Store at -20°C

Minimal Defined Media (M9)

This classic media recipe comes from Miller’s classic molecular biology text39.

Per Liter:

Na2HPO4 6 g

KH2PO4 3 g

NaCl 0.5 g

NH4Cl 1 g

AFTER autoclaving add:

0.01 M solution CaCl2 10 ml

1 M MgSO4∙H2O 1 ml

20% solution C source 10 ml

If vitamins or amino acids are needed:

Add to a final concentration of 1 µg/ml for vitamins

Add to a final concentration of 40 µg/ml for L-amino acids

Appendix G

GhoT Topology

GhoT amino acid sequence (without 6xhis):

MALFSKILIFYVIGVNISFVIIWFISHEKTHIRLLSAFLVGITWPMSLPVALLFSLF

Underlined residues are predicted to be part of transmembrane domain; Cytoplasmic; Non Cytoplasmic

Phobius analysis of transmembrane domains:

Appendix H

Sequence of pSU16R

TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGTTAATTA

AGAATTCACTAGTGGATCCTCTAGAGTCGACCTGCAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAAT

AACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTCGAGAAAAATAAACAAAAGAGTTTGTAGAAAC

GCAAAAAGGCCATCCGTCAGGATGGCCTTCTGCTTAATTTGATGCCTGGCAGTTTATGGCGGGCGTCCTGCCCGCCACCCTCCGGGCCGTTGCTTCGC

AACGTTCAAATCCGCTCCCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCC

TTTCGTTTTATTTGATGCCTGGCAGTTCCCTACTCTCGCATGGGGAGACCCCACACTACCATCGGCGCTACGGCGTTTCACTTCTGAGTTCGGCATGGG

GTCAGGTGGGACCACCGCGCTACTGCCGCCAGGCAAATTCTGTTTTATCAGACCGCTTCTGCGTTCTGATTTAATCTGTATCAGGCTGAAAATCTTCTC

TCATCCGCCAAAACAGCCAAGCTTTTACGCGAACGCGAAGTCCGACTCTAAGATGTCACGGAGGTTCAAGTTACCTTTAGCCGGAAGTGCTGGCATTT

TGTCCAATTGAGACTCGTGCAACTGGTCAGCGAACTGGTCGTAGAAATCAGCCAGTACATCACAAGACTCATATGTGTCAACCATAGTTTCGCGCACT

GCTTTGAACAGGTTCGCAGCGTCAGCCGGAATGGTACCGAAGGAGTCGTGAATCAGTGCAAAAGATTCGATTCCGTACTTCTCGTGTGCCCACACTAC

AGTCTTACGAAGGTGGCTACCGTCTTGGCTGTGTACAAAGTTAGGAGCGATACCAGACTCCTGTTTGTGTGCATCAATCTCGCTATCTTTGTTGGTGTT

AATGGTAGGCTGTAAGCGGAACTGACCGAGGAACATCAGGTTCAAGCGCGTCTGAATAGGCTTCTTGTATTCCTGCCACACAGGGAAACCATCAGGA

GTTACCCAATGCACAGCGCAACGCTTGCGAAGAATCTCTCCAGTCTTCTTATCTTTGACCTCAGCAGCCAGCAGCTTAGCAGCAGACTTAAGCCAGTT

CATTGCTTCAACCGCAGCTACCACCGTCACGCTCACAGATTCCCAAATCAGCTTAGCCATGTATCCAGCAGCCTGATTCGGCTGAGTGAACATCAGAC

CCTTGCCGGAATCAATAGCTGGCTGAATGGTATCTTCCAGCACTTGTTGACGGAAGCCGAACTCTTTGGACCCGTAAGCCAGCGTCATGACTGAACGC

TTAGTCACACTGCGAGTAACACCGTAAGCCAGCCATTGACCAGCCAGTGCCTTAGTGCCCAGCTTGACTTTCTCAGAGATTTCACCAGTGTTCTCATC

GGTCACGGTAACTACTTCGTTATCGGTCCCATTGATTGCGTCTGCTTGTAGAATCTCGTTGACTTTCTTAGCAACAATCCCGTAGATGTCCTGAACGGT

TTCACTAGGAAGCAAGTTAACCGCGCGACCACCTACCTCATCTCGGAGCATCGCGGAGAAGTGCTGGATGCCAGAGCAAGACCCGTCAAACGCCAGC

GGAAGGGAGCAGTTATAGCTCAGGCCGTGGTGCTGTACCCCAGCGTACTCAAAGCAGAACGCAAGGAAGCAGAACGGAGAATCTTGCTCAGCCCAC

CAAGTGTTCTCCAGTGGAGACTTAGCGCAAGCCATGATGTTCTCGTGGTTTTCCTCAATGAACTTGATGCGCTCAGGGAACGGAACCTTATCGACACC

CGCACAGTTTGCACCGTGGATTTTCAGCCAGTAGTAACCTTCCTTACCGATTGGTTTACCTTTCGCCAGCGTAAGCAGTCCTTTGGTCATATCGTTACC

TTGCGGGTTGAACATTGACACAGCGTAAACACGACCGCGCCAGTCCATGTTGTAAGGGAACCAGATGGCCTTATGGTTAGCAAACTTATTGGCTTGCT

CAAGCATGAACTCAAGGCTGATACGGCGAGACTTGCGAGCCTTGTCCTTGCGGTACACAGCAGCGGCAGCACGTTTCCACGCGGTGAGAGCCTCAGG

ATTCATGTCGATGTCTTCCGGTTTCATCGGGAGTTCTTCACGCTCAATCGCAGGGATGTCCTCGACCGGACAATGCTTCCACTTGGTGATTACGTTGGC

GACCGCTAGGACTTTCTTGTTGATTTTCCATGCGGTGTTTTGCGCAATGTTAATCGCTTTGTACACCTCAGGCATGTAAACGTCTTCGTAGCGCATCAG

TGCTTTCTTACTGTGAGTACGCACCAGCGCCAGAGGACGACGACCGTTAGCCCAATAGCCACCACCAGTAATGCCAGTCCACGGCTTAGGAGGAACT

ACGCAAGGTTGGAACATCGGAGAGATGCCAGCCAGCGCACCTGCACGGGTTGCGATAGCCTCAGCGTATTCAGGTGCGAGTTCGATAGTCTCAGAGT

CTTGACCTACTACGCCAGCATTTTGGCGGTGTAAGCTAACCATTCCGGTTGACTCAATGAGCATCTCGATGCAGCGTACTCCTACATGAATAGAGTCT

TCCTTATGCCACGAAGACCACGCCTCGCCACCGAGTAGACCCTTAGAGAGCATGTCAGCCTCGACAACTTGCATAAATGCTTTCTTGTAGACGTGCCC

TACGCGCTTGTTGAGTTGTTCCTCAACGTTTTTCTTGAAGTGCTTAGCTTCAAGGTCACGGATACGACCGAAGCGAGCCTCGTCCTCAATGGCCCGACC

GATTGCGCTTGCTACAGCCTGAACGGTTGTATTGTCAGCACTGGTTAGGCAAGCCAGAGTGGTCTTAATGGTGATGTACGCTACGGCTTCCGGCTTGA

TTTCTTGCAGGAACTGGAAGGCTGTCGGGCGCTTGCCGCGCTTAGCTTTCACTTCCTCAAACCAGTCGTTGATGCGTGCAATCATCTTAGGGAGTAGG

GTAGTGATGAGAGGCTTGGCGGCAGCGTTATCCGCAACCTCACCAGCTTTAAGTTGACGCTCAAACATCTTGCGGAAGCGTGCTTCACCCATCTCGTA

AGACTCATGCTCAAGGGCCAACTGTTCGCGAGCTAAACGCTCACCGTAATGGTCAGCCAGAGTGTTGAACGGGATAGCAGCCAGTTCGATGTCAGAG

AAGTCGTTCTTAGCGATGTTAATCGTGTTCATATGGTATCCTCTTGAGCTCGCTAGCCCAAAAAAACGGGTATGGAGAAACAGTAGAGAGTTGCGATA

AAAAGCGTCAGGTAGGATCCGCTAATCTTATGGATAAAAATGCTATGGCATAGCAAAGTGTGACGCCGTGCAAATAATCAATGTCCTAGGACTTTTC

TGCCGTGATTATAGACACTTTTGTTACGCGTTTTTGTCATGGCTTTGGTCCCGCTTTGTTACAGAATGCTTTTAATAAGCGGGGTTACCGGTTTGGTTAG

CGAGAAGAGCCAGTAAAAGACGCAGTGACGGCAATGTCTGATGCAATATGGACAATTGGTTTCTTCTCTGAATGGCGGGAGTATGAAAAGTATGGCT

GAAGCGCAAAATGATCCCCTGCTGCCGGGATACTCGTTTAATGCCCATCTGGTGGCGGGTTTAACGCCGATTGAGGCCAACGGTTATCTCGATTTTTT

TATCGACCGACCGCTGGGAATGAAAGGTTATATTCTCAATCTCACCATTCGCGGTCAGGGGGTGGTGAAAAATCAGGGACGAGAATTTGTTTGCCGA

CCGGGTGATATTTTGCTGTTCCCGCCAGGAGAGATTCATCACTACGGTCGTCATCCGGAGGCTCGCGAATGGTATCACCAGTGGGTTTACTTTCGTCC

GCGCGCCTACTGGCATGAATGGCTTAACTGGCCGTCAATATTTGCCAATACGGGGTTCTTTCGCCCGGATGAAGCGCACCAGCCGCATTTCAGCGACC

TGTTTGGGCAAATCATTAACGCCGGGCAAGGGGAAGGGCGCTATTCGGAGCTGCTGGCGATAAATCTGCTTGAGCAATTGTTACTGCGGCGCATGGA

AGCGATTAACGAGTCGCTCCATCCACCGATGGATAATCGGGTACGCGAGGCTTGTCAGTACATCAGCGATCACCTGGCAGACAGCAATTTTGATATC

GCCAGCGTCGCACAGCATGTTTGCTTGTCGCCGTCGCGTCTGTCACATCTTTTCCGCCAGCAGTTAGGGATTAGCGTCTTAAGCTGGCGCGAGGACCA

ACGTATCAGCCAGGCGAAGCTGCTTTTGAGCACCACCCGGATGCCTATCGCCACCGTCGGTCGCAATGTTGGTTTTGACGATCAACTCTATTTCTCGC

GGGTATTTAAAAAATGCACCGGGGCCAGCCCGAGCGAGTTCCGTGCCGGTTGTGAAGAAAAAGTGAATGATGTAGCCGTCAAGTTGTCATAATTGGT

AACGAATCAGACAATTGACGGCTTGACGGAGTAGCATAGGGTTTGCAGAATCCCTGCTTCGTCCAGATCTGGCACATTAGCATGTTGATTGTAACGAT

GACAGAGCGTTGCTGCCTGTGATCACCGCGGTTTCAAAATCGGCTCCGTCGATACTATGTTATACGCCAACTTTGAAAACAACTTTGAAAAAGCTGTT

TTCTGGTATTTAAGGTTTTAGAATGCAAGGAACAGTGAATTGGAGTTCGTCTTGTTATAATTAGCTTCTTGGGGTATCTTTAAATACTGTAGAAAAGA

GGAAGGAAATAATAAATGGCTAAAATGAGAATATCACCGGAATTGAAAAAACTGATCGAAAAATACCGCTGCGTAAAAGATACGGAAGGAATGTCT

CCTGCTAAGGTATATAAGCTGGTGGGAGAAAATGAAAACCTATATTTAAAAATGACGGACAGCCGGTATAAAGGGACCACCTATGATGTGGAACGG

GAAAAGGACATGATGCTATGGCTGGAAGGAAAGCTGCCTGTTCCAAAGGTCCTGCACTTTGAACGGCATGATGGCTGGAGCAATCTGCTCATGAGTG

AGGCCGATGGCGTCCTTTGCTCGGAAGAGTATGAAGATGAACAAAGCCCTGAAAAGATTATCGAGCTGTATGCGGAGTGCATCAGGCTCTTTCACTC

CATCGACATATCGGATTGTCCCTATACGAATAGCTTAGACAGCCGCTTAGCCGAATTGGATTACTTACTGAATAACGATCTGGCCGATGTGGATTGCG

AAAACTGGGAAGAAGACACTCCATTTAAAGATCCGCGCGAGCTGTATGATTTTTTAAAGACGGAAAAGCCCGAAGAGGAACTTGTCTTTTCCCACGG

CGACCTGGGAGACAGCAACATCTTTGTGAAAGATGGCAAAGTAAGTGGCTTTATTGATCTTGGGAGAAGCGGCAGGGCGGACAAGTGGTATGACATT

GCCTTCTGCGTCCGGTCGATCAGGGAGGATATCGGGGAAGAACAGTATGTCGAGCTATTTTTTGACTTACTGGGGATCAAGCCTGATTGGGAGAAAA

TAAAATATTATATTTTACTGGATGAATTGTTTTAGTACCTAGAATGCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCG

TAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTG

CCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCA

CCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTT

GGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGA

ACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGG

AGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCT

CGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGT

TATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGA

GGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATG

CCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGG

GCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAG

GCAGGGTGCCTTGATGTGGGCGCCGGCGGTCGAGTGGCGACGGCGCGGCTTGTCCGCGCCCTGGTAGATTGCCTGGCCGTAGGCCAGCCATTTTTGA

GCGGCCAGCGGCCGCGATAGGCCGACGCGAAGCGGCGGGGCGTAGGGAGCGCAGCGACCGAAGGGTAGGCGCTTTTTGCAGCTCTTCGGCTGTGCG

CTGGCCAGACAGTTATGCACAGGCCAGGCGGGTTTTAAGAGTTTTAATAAGTTTTAAAGAGTTTTAGGCGGAAAAATCGCCTTTTTTCTCTTTTATATC

AGTCACTTACATGTGTGACCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGGGTTCCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGT

GCTATCCACAGGAAAGAGACCTTTTCGACCTTTTTCCCCTGCTAGGGCAATTTGCCCTAGCATCTGCTCCGTACATTAGGAACCGGCGGATGCTTCGC

CCTCGATCAGGTTGCGGTAGCGCATGACTAGGATCGGGCCAGCCTGCCCCGCCTCCTCCTTCAAATCGTACTCCGGCAGGTCATTTGACCCGATCAGC

TTGCGCACGGTGAAACAGAACTTCTTGAACTCTCCGGCGCTGCCACTGCGTTCGTAGATCGTCTTGAACAACCATCTGGCTTCTGCCTTGCCTGCGGC

GCGGCGTGCCAGGCGGTAGAGAAAACGGCCGATGCCGGGATCGATCAAAAAGTAATCGGGGTGAACCGTCAGCACGTCCGGGTTCTTGCCTTCTGTG

ATCTCGCGGTACATCCAATCAGCTAGCTCGATCTCGATGTACTCCGGCCGCCCGGTTTCGCTCTTTACGATCTTGTAGCGGCTAATCAAGGCTTCACCC

TCGGATACCGTCACCAGGCGGCCGTTCTTGGCCTTCTTCGTACGCTGCATGGCAACGTGCGTGGTGTTTAACCGAATGCAGGTTTCTACCAGGTCGTCT

TTCTGCTTTCCGCCATCGGCTCGCCGGCAGAACTTGAGTACGTCCGCAACGTGTGGACGGAACACGCGGCCGGGCTTGTCTCCCTTCCCTTCCCGGTA

TCGGTTCATGGATTCGGTTAGATGGGAAACCGCCATCAGTACCAGGTCGTAATCCCACACACTGGCCATGCCGGCCGGCCCTGCGGAAACCTCTACGT

GCCCGTCTGGAAGCTCGTAGCGGATCACCTCGCCAGCTCGTCGGTCACGCTTCGACAGACGGAAAACGGCCACGTCCATGATGCTGCGACTATCGCG

GGTGCCCACGTCATAGAGCATCGGAACGAAAAAATCTGGTTGCTCGTCGCCCTTGGGCGGCTTCCTAATCGACGGCGCACCGGCTGCCGGCGGTTGC

CGGGATTCTTTGCGGATTCGATCAGCGGCCGCTTGCCACGATTCACCGGGGCGTGCTTCTGCCTCGATGCGTTGCCGCTGGGCGGCCTGCGCGGCCTT

CAACTTCTCCACCAGGTCATCACCCAGCGCCGCGCCGATTTGTACCGGGCCGGATGGTTTGCGACCGCTCACGCCGATTCCTCGGGCTTGGGGGTTCC

AGTGCCATTGCAGGGCCGGCAGACAACCCAGCCGCTTACGCCTGGCCAACCGCCCGTTCCTCCACACATGGGGCATTCCACGGCGTCGGTGCCTGGTT

GTTCTTGATTTTCCATGCCGCCTCCTTTAGCCGCTAAAATTCATCTACTCATTTATTCATTTGCTCATTTACTCTGGTAGCTGCGCGATGTATTCAGATA

GCAGCTCGGTAATGGTCTTGCCTTGGCGTACCGCGTACATCTTCAGCTTGGTGTGATCCTCCGCCGGCAACTGAAAGTTGACCCGCTTCATGGCTGGC

GTGTCTGCCAGGCTGGCCAACGTTGCAGCCTTGCTGCTGCGTGCGCTCGGACGGCCGGCACTTAGCGTGTTTGTGCTTTTGCTCATTTTCTCTTTACCT

CATTAACTCAAATGAGTTTTGATTTAATTTCAGCGGCCAGCGCCTGGACCTCGCGGGCAGCGTCGCCCTCGGGTTCTGATTCAAGAACGGTTGTGCCG

GCGGCGGCAGTGCCTGGGTAGCTCACGCGCTGCGTGATACGGGACTCAAGAATGGGCAGCTCGTACCCGGCCAGCGCCTCGGCAACCTCACCGCCGA

TGCGCGTGCCTTTGATCGCCCGCGACACGACAAAGGCCGCTTGTAGCCTTCCATCCGTGACCTCAATGCGCTGCTTAACCAGCTCCACCAGGTCGGCG

GTGGCCCATATGTCGTAAGGGCTTGGCTGCACCGGAATCAGCACGAAGTCGGCTGCCTTGATCGCGGACACAGCCAAGTCCGCCGCCTGGGGCGCTC

CGTCGATCACTACGAAGTCGCGCCGGCCGATGGCCTTCACGTCGCGGTCAATCGTCGGGCGGTCGATGCCGACAACGGTTAGCGGTTGATCTTCCCGC

ACGGCCGCCCAATCGCGGGCACTGCCCTGGGGATCGGAATCGACTAACAGAACATCGGCCCCGGCGAGTTGCAGGGCGCGGGCTAGATGGGTTGCG

ATGGTCGTCTTGCCTGACCCGCCTTTCTGGTTAAGTACAGCGATAACCTTCATGCGTTCCCCTTGCGTATTTGTTTATTTACTCATCGCATCATATACGC

AGCGACCGCATGACGCAAGCTGTTTTACTCAAATACACATCACCTTTTTAGACGGCGGCGCTCGGTTTCTTCAGCGGCCAAGCTGGCCGGCCAGGCCG

CCAGCTTGGCATCAGACAAACCGGCCAGGATTTCATGCAGCCGCACGGTTGAGACGTGCGCGGGCGGCTCGAACACGTACCCGGCCGCGATCATCTC

CGCCTCGATCTCTTCGGTAATGAAAAACGGTTCGTCCTGGCCCATTCGCCAATCCGGAT

Appendix I

Sequence of pSU13R

GCATCGACCGCCCGACGATTGACCGCGACGTGAAGGCCATCGGCCGGCGCGACTTCGTAGTGATCGACGGAGCGCCCCAGGCGGCGGACTTGGCTGT

GTCCGCGATCAAGGCAGCCGACTTCGTGCTGATTCCGGTGCAGCCAAGCCCTTACGACATATGGGCCACCGCCGACCTGGTGGAGCTGGTTAAGCAG

CGCATTGAGGTCACGGATGGAAGGCTACAAGCGGCCTTTGTCGTGTCGCGGGCGATCAAAGGCACGCGCATCGGCGGTGAGGTTGCCGAGGCGCTGG

CCGGGTACGAGCTGCCCATTCTTGAGTCCCGTATCACGCAGCGCGTGAGCTACCCAGGCACTGCCGCCGCCGGCACAACCGTTCTTGAATCAGAACCC

GAGGGCGACGCTGCCCGCGAGGTCCAGGCGCTGGCCGCTGAAATTAAATCAAAACTCATTTGAGTTAATGAGGTAAAGAGAAAATGAGCAAAAGCA

CAAACACGCTAAGTGCCGGCCGTCCGAGCGCACGCAGCAGCAAGGCTGCAACGTTGGCCAGCCTGGCAGACACGCCAGCCATGAAGCGGGTCAACT

TTCAGTTGCCGGCGGAGGATCACACCAAGCTGAAGATGTACGCGGTACGCCAAGGCAAGACCATTACCGAGCTGCTATCTGAATACATCGCGCAGCT

ACCAGAGTAAATGAGCAAATGAATAAATGAGTAGATGAATTTTAGCGGCTAAAGGAGGCGGCATGGAAAATCAAGAACAACCAGGCACCGACGCCG

TGGAATGCCCCATGTGTGGAGGAACGGGCGGTTGGCCAGGCGTAAGCGGCTGGGTTGTCTGCCGGCCCTGCAATGGCACTGGAACCCCCAAGCCCGA

GGAATCGGCGTGAGCGGTCGCAAACCATCCGGCCCGGTACAAATCGGCGCGGCGCTGGGTGATGACCTGGTGGAGAAGTTGAAGGCCGCGCAGGCC

GCCCAGCGGCAACGCATCGAGGCAGAAGCACGCCCCGGTGAATCGTGGCAAGCGGCCGCTGATCGAATCCGCAAAGAATCCCGGCAACCGCCGGCA

GCCGGTGCGCCGTCGATTAGGAAGCCGCCCAAGGGCGACGAGCAACCAGATTTTTTCGTTCCGATGCTCTATGACGTGGGCACCCGCGATAGTCGCA

GCATCATGGACGTGGCCGTTTTCCGTCTGTCGAAGCGTGACCGACGAGCTGGCGAGGTGATCCGCTACGAGCTTCCAGACGGGCACGTAGAGGTTTC

CGCAGGGCCGGCCGGCATGGCCAGTGTGTGGGATTACGACCTGGTACTGATGGCGGTTTCCCATCTAACCGAATCCATGAACCGATACCGGGAAGGG

AAGGGAGACAAGCCCGGCCGCGTGTTCCGTCCACACGTTGCGGACGTACTCAAGTTCTGCCGGCGAGCCGATGGCGGAAAGCAGAAAGACGACCTG

GTAGAAACCTGCATTCGGTTAAACACCACGCACGTTGCCATGCAGCGTACGAAGAAGGCCAAGAACGGCCGCCTGGTGACGGTATCCGAGGGTGAA

GCCTTGATTAGCCGCTACAAGATCGTAAAGAGCGAAACCGGGCGGCCGGAGTACATCGAGATCGAGCTAGCTGATTGGATGTACCGCGAGATCACA

GAAGGCAAGAACCCGGACGTGCTGACGGTTCACCCCGATTACTTTTTGATCGATCCCGGCATCGGCCGTTTTCTCTACCGCCTGGCACGCCGCGCCGC

AGGCAAGGCAGAAGCCAGATGGTTGTTCAAGACGATCTACGAACGCAGTGGCAGCGCCGGAGAGTTCAAGAAGTTCTGTTTCACCGTGCGCAAGCTG

ATCGGGTCAAATGACCTGCCGGAGTACGATTTGAAGGAGGAGGCGGGGCAGGCTGGCCCGATCCTAGTCATGCGCTACCGCAACCTGATCGAGGGC

GAAGCATCCGCCGGTTCCTAATGTACGGAGCAGATGCTAGGGCAAATTGCCCTAGCAGGGGAAAAAGGTCGAAAAGGTCTCTTTCCTGTGGATAGCA

CGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGAACCCGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGTCACACATGTAAGTGAC

TGATATAAAAGAGAAAAAAGGCGATTTTTCCGCCTAAAACTCTTTAAAACTTATTAAAACTCTTAAAACCCGCCTGGCCTGTGCATAACTGTCTGGCC

AGCGCACAGCCGAAGAGCTGCAAAAAGCGCCTACCCTTCGGTCGCTGCGCTCCCTACGCCCCGCCGCTTCGCGTCGGCCTATCGCGGCCGCTGGCCG

CTCAAAAATGGCTGGCCTACGGCCAGGCAATCTACCAGGGCGCGGACAAGCCGCGCCGTCGCCACTCGACCGCCGGCGCCCACATCAAGGCACCCTG

CCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAA

GCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGC

GGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCT

TCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGA

TAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCC

TGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTG

CGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTC

AGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTC

CAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAG

TGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATC

CGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTT

CTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGCATTCTAGGTACTAAAACAATTCATCCAGTAAAATATAATAT

TTTATTTTCTCCCAATCAGGCTTGATCCCCAGTAAGTCAAAAAATAGCTCGACATACTGTTCTTCCCCGATATCCTCCCTGATCGACCGGACGCAGAAG

GCAATGTCATACCACTTGTCCGCCCTGCCGCTTCTCCCAAGATCAATAAAGCCACTTACTTTGCCATCTTTCACAAAGATGTTGCTGTCTCCCAGGTCG

CCGTGGGAAAAGACAAGTTCCTCTTCGGGCTTTTCCGTCTTTAAAAAATCATACAGCTCGCGCGGATCTTTAAATGGAGTGTCTTCTTCCCAGTTTTCG

CAATCCACATCGGCCAGATCGTTATTCAGTAAGTAATCCAATTCGGCTAAGCGGCTGTCTAAGCTATTCGTATAGGGACAATCCGATATGTCGATGGA

GTGAAAGAGCCTGATGCACTCCGCATACAGCTCGATAATCTTTTCAGGGCTTTGTTCATCTTCATACTCTTCCGAGCAAAGGACGCCATCGGCCTCAC

TCATGAGCAGATTGCTCCAGCCATCATGCCGTTCAAAGTGCAGGACCTTTGGAACAGGCAGCTTTCCTTCCAGCCATAGCATCATGTCCTTTTCCCGTT

CCACATCATAGGTGGTCCCTTTATACCGGCTGTCCGTCATTTTTAAATATAGGTTTTCATTTTCTCCCACCAGCTTATATACCTTAGCAGGAGACATTCC

TTCCGTATCTTTTACGCAGCGGTATTTTTCGATCAGTTTTTTCAATTCCGGTGATATTCTCATTTTAGCCATTTATTATTTCCTTCCTCTTTTCTACAGTA

TTTAAAGATACCCCAAGAAGCTAATTATAACAAGACGAACTCCAATTCACTGTTCCTTGCATTCTAAAACCTTAAATACCAGAAAACAGCTTTTTCAA

AGTTGTTTTCAAAGTTGGCGTATAACATAGTATCGACGGAGCCGATTTTGAAACCGCGGTGATCACAGGCAGCAACGCTCTGTCATCGTTACAATCAA

CATGCTAATGTGCCTGTCAAATGGACGAAGCAGGGATTCTGCAAACCCTATGCTACTCCGTCAAGCCGTCAATTGTCTGATTCGTGCGAAGCAACGGC

CCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAAC

TCTTTTGTTTATTTTTCTCGAGTTCCTCCTTTCAGCAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGTTATTGCTCAGCGGTG

GCAGCAGCCAACTCAGCTTCCTTTCGGGCTTTGTTAGCAGCCGGATCTGCAGGTCGACTCTAGAGGATCCACTAGTGAATTCTTAATTAACCATGGTA

TATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGGGAATTGTTATCCGCTCACAATTCCCCTATAGTGAGTCGTATTAATCCGGATTGGCGAAT

GGGCCAGGACGAACCGTTTTTCATTACCGAAGAGATCGAGGCGGAGATGATCGCGGCCGGGTACGTGTTCGAGCCGCCCGCGCACGTCTCAACCGTG

CGGCTGCATGAAATCCTGGCCGGTTTGTCTGATGCCAAGCTGGCGGCCTGGCCGGCCAGCTTGGCCGCTGAAGAAACCGAGCGCCGCCGTCTAAAAA

GGTGATGTGTATTTGAGTAAAACAGCTTGCGTCATGCGGTCGCTGCGTATATGATGCGATGAGTAAATAAACAAATACGCAAGGGGAACGCATGAAG

GTTATCGCTGTACTTAACCAGAAAGGCGGGTCAGGCAAGACGACCATCGCAACCCATCTAGCCCGCGCCCTGCAACTCGCCGGGGCCGATGTTCTGT

TAGTCGATTCCGATCCCCAGGGCAGTGCCCGCGATTGGGCGGCCGTGCGGGAAGATCAACCGCTAACCGTTGTCG

Appendix J

Gel Electrophoresis Tips

• Do not Overload DNA

• Don not use 50x TAE by accident

• Annotate Gels in PPT

• Conserve GelRed

• %Agarose

• Voltage

pSU13

digest

Uncut AP3 AP3 AP3B Barnase

5kb

600

300

4kb

(+)(-) 1 2 3 64 5 7 8 9 10

800

936

400

Pick ONE

Figure 1. 50 ml gel, <4.0ul GelRed 100V for 5min 85V for 90min 1ul of each ladder (NEB 1kb, GeneRuler)

Figure 2. 30 ml gel (colony PCR), <2.5ul GelRed 90V for 60min 0.75ul GeneRuler 100bp plus DNA Ladder

BIBLIOGRAPHY

1. Young JM, Kuykendall LD, Martínez-Romero E, Kerr A, Sawada H. A revision of Rhizobium

Frank 1889, with an emended description of the genus, and the inclusion of all species of

Agrobacterium Conn 1942 and Allorhizobium undicola de Lajudie et al. 1998 as new

combinations: Rhizobium radiobacter, R. rhizogenes, R. rubi,. Int J Syst Evol Microbiol.

2001;51(Pt 1):89-103. doi:10.1099/00207713-51-1-89.

2. Engström P, Zambryski P, Van Montagu M, Stachel S. Characterization of Agrobacterium

tumefaciens virulence proteins induced by the plant factor acetosyringone. J Mol Biol.

1987;197(4):635-645. doi:10.1016/0022-2836(87)90470-0.

3. Cangelosi G a, Ankenbauer RG, Nester EW. Sugars induce the Agrobacterium virulence genes

through a periplasmic binding protein and a transmembrane signal protein. Proc Natl Acad Sci U S

A. 1990;87(17):6708-6712. doi:10.1073/pnas.87.17.6708.

4. Morris JW, Morris RO. Identification of an Agrobacterium tumefaciens virulence gene inducer

from the pinaceous gymnosperm Pseudotsuga menziesii. Proc Natl Acad Sci U S A.

1990;87(9):3614-3618. doi:10.1073/pnas.87.9.3614.

5. Hiei Y, Komari T. Agrobacterium-mediated transformation of rice using immature embryos or

calli induced from mature seed. Nat Protoc. 2008;3(5):824-834. doi:10.1038/nprot.2008.46.

6. Wood DW, Setubal JC, Kaul R, et al. The genome of the natural genetic engineer Agrobacterium

tumefaciens C58. Science. 2001;294(5550):2317-2323. doi:10.1126/science.1066804.

7. Goodner B, Hinkle G, Gattung S, et al. Genome sequence of the plant pathogen and biotechnology

agent Agrobacterium tumefaciens C58. Science. 2001;294(5550):2323-2328.

doi:10.1126/science.1066803.

8. Chetty VJ, Ceballos N, Garcia D, Narváez-Vásquez J, Lopez W, Orozco-Cárdenas ML.

Evaluation of four Agrobacterium tumefaciens strains for the genetic transformation of tomato

(Solanum lycopersicum L.) cultivar Micro-Tom. Plant Cell Rep. 2013;32(2):239-247.

doi:10.1007/s00299-012-1358-1.

9. Barton KA, Binns AN, Matzke AJM, Chilton M-D. Regeneration of intact tobacco plants

containing full length copies of genetically engineered T-DNA, and transmission of T-DNA to R1

progeny. Cell. 1983;32(4):1033-1043. doi:10.1016/0092-8674(83)90288-X.

10. Hood EE, Jen G, Kayes L, Kramer J, Fraley RT, Chilton M-D. Restriction Endonuclease Map of

pTi Bo542, a Potential Ti Plasmid Vector for Genetic Engineering of Plants. Bio/Technology.

1984;2(8):702-709. doi:10.1038/nbt0884-702.

11. Hood EE, Helmer GL, Fraley RT, Chilton MD. The hypervirulence of Agrobacterium tumefaciens

A281 is encoded in a region of pTiBo542 outside of T-DNA. J Bacteriol. 1986;168(3):1291-1301.

12. Hood EE, Gelvin SB, Melchers LS, Hoekema A. New Agrobacterium helper plasmids for gene

transfer to plants. Transgenic Res. 1993;2(4):208-218. doi:10.1007/BF01977351.

13. Darbani B, Farajnia S, Toorchi M, Zakerbostanabad S, Noeparvar S, Neal Stewart Jr. C. DNA-

Delivery Methods to Produce Transgenic Plants. Biotechnology(Faisalabad). 2008;7(3):385-402.

doi:10.3923/biotech.2008.385.402.

14. Wu H-Y, Liu K-H, Wang Y-C, et al. AGROBEST: an efficient Agrobacterium-mediated transient

expression method for versatile gene function analyses in Arabidopsis seedlings. Plant Methods.

2014;10(1):1-16. doi:10.1186/1746-4811-10-19.

15. Kumar SV, Misquitta RW, Reddy VS, Rao BJ, Rajam MV. Genetic transformation of the green

alga--Chlamydomonas reinhardtii by Agrobacterium tumefaciens. Plant Sci. 2004;166(3):731-738.

doi:10.1016/j.plantsci.2003.11.012.

16. de Groot MJ, Bundock P, Hooykaas PJ, Beijersbergen a G. Agrobacterium tumefaciens-mediated

transformation of filamentous fungi. Nat Biotechnol. 1998;16(9):839-842. doi:10.1038/nbt0998-

839.

17. Gomord V, Fitchette AC, Menu-Bouaouiche L, et al. Plant-specific glycosylation patterns in the

context of therapeutic protein production. Plant Biotechnol J. 2010;8(5):564-587.

doi:10.1111/j.1467-7652.2009.00497.x.

18. Kolotilin I, Topp E, Cox E, et al. Plant-based solutions for veterinary immunotherapeutics and

prophylactics. Vet Res. 2014;45(1):1-12. doi:10.1186/s13567-014-0117-4.

19. Doyle IMP, Dale G, Choi H, City B. (12) United States Patent. 2012;2(12).

20. Hsiao, Bacani, Carvalho, Curtis. Development of a low capital investment reactor system:

application for plant cell suspension culture. Biotechnol Prog. 1999;15(1):114-122.

doi:10.1021/bp980103+.

21. Florez SL, Curtis MS, Shaw SE, Hamaker NK, Larsen JS, Curtis WR. A Temporary Immersion

Plant Propagation Bioreactor with Decoupled Gas and Liquid Flows for Enhanced Control of Gas

Phase. Biotechnol Prog. 2015:n/a - n/a. doi:10.1002/btpr.2221.

22. Morales VM, Sequeira L. Suicide vector for transposon mutagenesis in Pseudomonas

solanacearum. J Bacteriol. 1985;163(3):1263-1264.

23. Collens JI, Lee DR, Seeman AM, Curtis WR. Development of Auxotrophic Agrobacterium

tumefaciens for Gene Transfer in Plant Tissue Culture. Biotechnol Prog. 2004;20(3):890-896.

doi:10.1021/bp034306w.

24. Lippincott BB, Lippincott JA. Characteristics of Agrobacterium tumefaciens auxotrophic mutant

infectivity. J Bacteriol. 1966;92(4):937-945. http://www.ncbi.nlm.nih.gov/pubmed/5926761.

25. Scott HN, Laible PD, Hanson DK. Sequences of versatile broad-host-range vectors of the RK2

family. Plasmid. 2003;50(1):74-79. doi:10.1016/S0147-619X(03)00030-1.

26. Heeb S, Itoh Y, Nishijyo T, et al. Small, stable shuttle vectors based on the minimal pVS1 replicon

for use in gram-negative, plant-associated bacteria. Mol Plant Microbe Interact. 2000;13(2):232-

237. doi:10.1094/MPMI.2000.13.2.232.

27. Stenzel TT, Patel P, Bastia D. The integration host factor of Escherichia coli binds to bent DNA at

the origin of replication of the plasmid pSC101. Cell. 1987;49(5):709-717. doi:10.1016/0092-

8674(87)90547-2.

28. Shafferman A, Helinski DR. Structural properties of the beta origin of replication of plasmid R6K.

J Biol Chem. 1983;258(7):4083-4090. http://www.ncbi.nlm.nih.gov/pubmed/6300075.

29. Hershfield V, Boyer HW, Yanofsky C, Lovett MA, Helinski DR. Plasmid ColEl as a molecular

vehicle for cloning and amplification of DNA. Proc Natl Acad Sci U S A. 1974;71(9):3455-3459.

doi:10.1073/pnas.71.9.3455.

30. Tomizawa J. Control of ColE1 plasmid replication: the process of binding of RNA I to the primer

transcript. Cell. 1984;38(3):861-870. http://www.ncbi.nlm.nih.gov/pubmed/6207934.

31. Hajdukiewicz P, Svab Z, Maliga P. The small, versatile pPZP family of Agrobacterium binary

vectors for plant transformation. Plant Mol Biol. 1994;25(6):989-994. doi:10.1007/BF00014672.

32. Davison J. Genetic tools for pseudomonads, rhizobia, and other gram-negative bacteria.

Biotechniques. 2002;32(2):386-388, 390, 392-394, passim.

http://www.ncbi.nlm.nih.gov/pubmed/11848415.

33. Gallegos MT, Schleif R, Bairoch A, Hofmann K, Ramos JL. Arac/XylS family of transcriptional

regulators. Microbiol Mol Biol Rev. 1997;61(4):393-410.

http://www.ncbi.nlm.nih.gov/pubmed/9409145.

34. Weickert MJ, Adhya S. A family of bacterial regulators homologous to Gal and Lac repressors. J

Biol Chem. 1992;267(22):15869-15874. http://www.ncbi.nlm.nih.gov/pubmed/1639817.

35. Remaut E, Stanssens P, Fiers W. Plasmid vectors for high-efficiency expression controlled by the

PL promoter of coliphage lambda. Gene. 1981;15(1):81-93.

http://www.ncbi.nlm.nih.gov/pubmed/6271633.

36. Okorokov a L, Hartley RW, Panov KI. An improved system for ribonuclease Ba expression.

Protein Expr Purif. 1994;5(6):547-552. doi:10.1006/prep.1994.1075.

37. Saïda F, Uzan M, Odaert B, Bontems F. Expression of highly toxic genes in E. coli: special

strategies and genetic tools. Curr Protein Pept Sci. 2006;7(1):47-56.

doi:10.2174/138920306775474095.

38. Giacalone MJ, Gentile AM, Lovitt BT, Berkley NL, Gunderson CW, Surber MW. Toxic protein

expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter

system. Biotechniques. 2006;40(3):355-364. doi:10.2144/000112112.

39. Miller J. Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor

Laboratory Press; 1972.

40. Deutscher J. The mechanisms of carbon catabolite repression in bacteria. Curr Opin Microbiol.

2008;11(2):87-93. doi:10.1016/j.mib.2008.02.007.

41. Noel RJ, Reznikoff WS. Structural studies of lacUV5-RNA polymerase interactions in vitro.

Ethylation interference and missing nucleoside analysis. J Biol Chem. 2000;275(11):7708-7712.

http://www.ncbi.nlm.nih.gov/pubmed/10713082.

42. Schleif R. AraC protein, regulation of the l-arabinose operon in Escherichia coli, and the light

switch mechanism of AraC action. FEMS Microbiol Rev. 2010;34(5):779-796.

doi:10.1111/j.1574-6976.2010.00226.x.

43. Kemner JM, Liang X, Nester EW. The Agrobacterium tumefaciens virulence gene chvE is part of

a putative ABC-type sugar transport operon. J Bacteriol. 1997;179(7):2452-2458.

44. Zhao J, Binns AN. Characterization of the mmsAB-araD1 (gguABC) genes of Agrobacterium

tumefaciens. J Bacteriol. 2011;193(23):6586-6596. doi:10.1128/JB.05790-11.

45. Chen CY, Winans SC. Controlled expression of the transcriptional activator gene virG in

Agrobacterium tumefaciens by using the Escherichia coli lac promoter. J Bacteriol.

1991;173(3):1139-1144.

46. Newman JR, Fuqua C. Broad-host-range expression vectors that carry the L-arabinose-inducible

Escherichia coli araBAD promoter and the araC regulator. Gene. 1999;227(2):197-203.

doi:10.1016/S0378-1119(98)00601-5.

47. Studier FW, Moffatt B a. Use of bacteriophage T7 RNA polymerase to direct selective high-level

expression of cloned genes. J Mol Biol. 1986;189(1):113-130. doi:10.1016/0022-2836(86)90385-

2.

48. Davanloo P, Rosenberg AH, Dunn JJ, Studier FW. Cloning and expression of the gene for

bacteriophage T7 RNA polymerase. Proc Natl Acad Sci. 1984;81(7):2035-2039.

doi:10.1073/pnas.81.7.2035.

49. Olins PO, Rangwala SH. A novel sequence element derived from bacteriophage T7 mRNA acts as

an enhancer of translation of the lacZ gene in Escherichia coli. J Biol Chem. 1989;264(29):16973-

16976. http://www.ncbi.nlm.nih.gov/pubmed/2676996.

50. Wycuff DR, Matthews KS. Generation of an AraC-araBAD promoter-regulated T7 expression

system. Anal Biochem. 2000;277(1):67-73. doi:10.1006/abio.1999.4385.

51. Shis DL, Bennett MR. Library of synthetic transcriptional AND gates built with split T7 RNA

polymerase mutants. Proc Natl Acad Sci U S A. 2013;110(13):5028-5033.

doi:10.1073/pnas.1220157110.

52. Kang Y, Son MS, Hoang TT. One step engineering of T7-expression strains for protein

production: increasing the host-range of the T7-expression system. Protein Expr Purif.

2007;55(2):325-333. doi:10.1016/j.pep.2007.06.014.

53. Zhu L, Kaback HR, Dalbey RE. YidC protein, a molecular chaperone for LacY protein folding via

the SecYEG protein machinery. J Biol Chem. 2013;288(39):28180-28194.

doi:10.1074/jbc.M113.491613.

54. Van Melderen L, Saavedra De Bast M. Bacterial toxin-antitoxin systems: more than selfish

entities? PLoS Genet. 2009;5(3):e1000437. doi:10.1371/journal.pgen.1000437.

55. Dao-Thi M-H, Van Melderen L, De Genst E, et al. Molecular basis of gyrase poisoning by the

addiction toxin CcdB. J Mol Biol. 2005;348(5):1091-1102. doi:10.1016/j.jmb.2005.03.049.

56. Yamamoto S, Kiyokawa K, Tanaka K, Moriguchi K, Suzuki K. Novel toxin-antitoxin system

composed of serine protease and AAA-ATPase homologues determines the high level of stability

and incompatibility of the tumor-inducing plasmid pTiC58. J Bacteriol. 2009;191(14):4656-4666.

doi:10.1128/JB.00124-09.

57. Kussell E, Kishony R, Balaban NQ, Leibler S. Bacterial persistence: A model of survival in

changing environments. Genetics. 2005;169(4):1807-1814. doi:10.1534/genetics.104.035352.

58. Benz J, Meinhart A. Antibacterial effector/immunity systems: It’s just the tip of the iceberg. Curr

Opin Microbiol. 2014;17(1):1-10. doi:10.1016/j.mib.2013.11.002.

59. Wang X, Lord DM, Cheng H-Y, et al. A new type V toxin-antitoxin system where mRNA for

toxin GhoT is cleaved by antitoxin GhoS. Nat Chem Biol. 2012;8(10):855-861.

doi:10.1038/nchembio.1062.

60. Cheng HY, Soo VWC, Islam S, McAnulty MJ, Benedik MJ, Wood TK. Toxin GhoT of the

GhoT/GhoS toxin/antitoxin system damages the cell membrane to reduce adenosine triphosphate

and to reduce growth under stress. Environ Microbiol. 2014;16(6):1741-1754. doi:10.1111/1462-

2920.12373.

61. Guillet V, Lapthorn A, Hartley R, Mauguen Y. Recognition between a bacterial ribonuclease,

barnase, and its natural inhibitor, barstar. Structure. 1993;1(3):165-176. doi:10.1016/0969-

2126(93)90018-C.

62. Ramos HJO, Souza EM, Soares-Ramos JRL, Pedrosa FO. Antibiosis by Bacillus

amyloliquefaciens ribonuclease barnase expressed in Escherichia coli against symbiotic and

endophytic nitrogen-fixing bacteria. J Biotechnol. 2006;126(3):291-294.

doi:10.1016/j.jbiotec.2006.04.020.

63. Wang L, Schultz PG. A general approach for the generation of orthogonal tRNAs. Chem Biol.

2001;8(9):883-890. http://www.ncbi.nlm.nih.gov/pubmed/11564556.

64. Burgess DG, Ralston EJ, Hanson WG, et al. A novel, two-component system for cell lethality and

its use in engineering nuclear male-sterility in plants. Plant J. 2002;31(1):113-125.

http://www.ncbi.nlm.nih.gov/pubmed/12100487.

65. Ma LS, Hachani A, Lin JS, Filloux A, Lai EM. Agrobacterium tumefaciens deploys a superfamily

of type VI secretion DNase effectors as weapons for interbacterial competition in planta. Cell Host

Microbe. 2014;16(1):94-104. doi:10.1016/j.chom.2014.06.002.

66. Hood RD, Singh P, Hsu F, et al. A type VI secretion system of Pseudomonas aeruginosa targets a

toxin to bacteria. Cell Host Microbe. 2010;7(1):25-37. doi:10.1016/j.chom.2009.12.007.

67. Russell AB, Hood RD, Bui NK, LeRoux M, Vollmer W, Mougous JD. Type VI secretion delivers

bacteriolytic effectors to target cells. Nature. 2011;475(7356):343-347. doi:10.1038/nature10244.

68. Russell AB, Singh P, Brittnacher M, et al. A widespread bacterial type VI secretion effector

superfamily identified using a heuristic approach. Cell Host Microbe. 2012;11(5):538-549.

doi:10.1016/j.chom.2012.04.007.

69. Benz J, Sendlmeier C, Barends TRM, Meinhart A. Structural insights into the effector - immunity

system Tse1/Tsi1 from Pseudomonas aeruginosa. PLoS One. 2012;7(7).

doi:10.1371/journal.pone.0040453.

70. Reyrat JM, Pelicic V, Gicquel B, Rappuoli R. Counterselectable markers: untapped tools for

bacterial genetics and pathogenesis. Infect Immun. 1998;66(9):4011-4017.

http://www.ncbi.nlm.nih.gov/pubmed/9712740.

71. Luo P, He X, Liu Q, Hu C. Developing Universal Genetic Tools for Rapid and Efficient Deletion

Mutation in Vibrio Species Based on Suicide T-Vectors Carrying a Novel Counterselectable

Marker, vmi480. Isalan M, ed. PLoS One. 2015;10(12):e0144465.

doi:10.1371/journal.pone.0144465.

72. Gay P, Le Coq D, Steinmetz M, Berkelman T, Kado CI. Positive selection procedure for

entrapment of insertion sequence elements in gram-negative bacteria. J Bacteriol.

1985;164(2):918-921. http://www.ncbi.nlm.nih.gov/pubmed/2997137.

73. Ma Y, Weiss LM, Huang H. Inducible suicide vector systems for Trypanosoma cruzi. Microbes

Infect. 2015;17(6):440-450. doi:10.1016/j.micinf.2015.04.003.

74. Bej AK, Perlin MH, Atlas RM. Model suicide vector for containment of genetically engineered

microorganisms. Appl Environ Microbiol. 1988;54(10):2472-2477.

75. Contreras A, Molin S, Ramos JL. Conditional-suicide containment system for bacteria which

mineralize aromatics. Appl Environ Microbiol. 1991;57(5):1504-1508.

76. Recorbet G, Robert C, Givaudan A, Kudla B, Normand P, Faurie G. Conditional suicide system of

Escherichia coli released into soil that uses the Bacillus subtilis sacB gene. Appl Environ

Microbiol. 1993;59(5):1361-1366. http://www.ncbi.nlm.nih.gov/pubmed/8517732.

77. Green M, Sambrook J. Molecular Cloning: A Laboratory Manual. Fourth. Cold Spring Harbor,

NY: Cold Spring Harbor Laboratory Press; 2012.

78. Myers J a, Curtis BS, Curtis WR. Improving accuracy of cell and chromophore concentration

measurements using optical density. BMC Biophys. 2013;6(1):4. doi:10.1186/2046-1682-6-4.

79. Reese MG. Application of a time-delay neural network to promoter annotation in the Drosophila

melanogaster genome. Comput Chem. 2001;26(1):51-56.

http://www.ncbi.nlm.nih.gov/pubmed/11765852.

80. Chapman KA, Burgess RR. Construction of bacteriophage T7 late promoters with point mutations

and characterization by in vitro transcription properties. Nucleic Acids Res. 1987;15(13):5413-

5432. doi:10.1093/nar/15.13.5413.

81. Sousa R, Chung YJ, Rose JP, Wang BC. Crystal structure of bacteriophage T7 RNA polymerase at

3.3 A resolution. Nature. 1993;364(6438):593-599. doi:10.1038/364593a0.

82. Studier FW. Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J

Mol Biol. 1991;219(1):37-44. doi:10.1016/0022-2836(91)90855-Z.

83. Spronk C a. EM, Bonvin a. MJJ, Radha PK, Melacini G, Boelens R, Kaptein R. The solution

structure of lac repressor headpeice 62 complexed to a symmetrical lac operator sequence

determined by NMR and restrained molecular dynamics. Structure. 1999;7:1483-1492.

84. Soisson SM, MacDougall-Shackleton B, Schleif R, Wolberger C. The 1.6 A crystal structure of

the AraC sugar-binding and dimerization domain complexed with D-fucose. J Mol Biol.

1997;273(1):226-237. doi:10.1006/jmbi.1997.1314.

85. Carter AD, Morris CE, McAllister WT. Revised transcription map of the late region of

bacteriophage T7 DNA. J Virol. 1981;37(2):636-642.

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=171051&tool=pmcentrez&rendertype

=abstract.

86. Mairhofer J, Scharl T, Marisch K, Cserjan-Puschmann M, Striedner G. Comparative transcription

profiling and in-depth characterization of plasmid-based and plasmid-free Escherichia coli

expression systems under production conditions. Appl Environ Microbiol. 2013;79(12):3802-3812.

doi:10.1128/AEM.00365-13.

87. Mairhofer J, Wittwer A, Cserjan-Puschmann M, Striedner G. Preventing T7 RNA polymerase

read-through transcription-A synthetic termination signal capable of improving bioprocess

stability. ACS Synth Biol. 2015;4(3):265-273. doi:10.1021/sb5000115.

88. Pan SQ, Jin S, Boulton MI, Hawes M, Gordon MP, Nester EW. An Agrobacterium virulence

factor encoded by a Ti plasmid gene or a chromosomal gene is required for T-DNA transfer into

plants. Mol Microbiol. 1995;17(2):259-269. doi:10.1111/j.1365-2958.1995.mmi_17020259.x.

89. Huang MLW, Cangelosi GA, Halperin W, Nester EW. A chromosomal Agrobacterium

tumefaciens gene required for effective plant signal transduction. J Bacteriol. 1990;172(4):1814-

1822.

90. Gibson DG, Young L, Chuang R, Venter JC, Hutchison C a, Smith HO. Enzymatic assembly of

DNA molecules up to several hundred kilobases. Nat Methods. 2009;6(5):343-345.

Academic Vitae

Nathaniel Hamaker nkh5051@psu.edu

EDUCATION

The Pennsylvania State University – University Park, PA

Schreyer Honors College

B.S. Chemical Engineering, Expected May 2016

B.S. Plant Biology, Expected May 2016

Study Abroad: Costa Rica, January 2013

EXPERIENCE

Independent Research in the Lab of Dr. Wayne Curtis – University Park, PA September 2012 – Present

Characterized oxygen transfer limitation on hairy root cultures

Designed CRISPR-Cas9 targets for the knockout of specific genes in a plant of interest

Developed techniques for the propagation of a recalcitrant plant species (Crocus sativus)

Assembled contiguous DNA fragments into a genome scaffold using Mauve alignment software and

optical restriction mapping

Annotated a completed bacterial genome with RAST in search of genes potentially involved in algal

symbiosis as well as for use in comparative genomics

Working with new lab members to introduce project goals and teach biomolecular methods

Teaching Assistant for Plant Biology Course – University Park, PA August – December 2015

Taught the laboratory component of the course to solidify concepts and introduce necessary techniques of

biology research

Fostered students’ ability to think critically through development of experimental plans and research ideas

Responsible for grading my section of 18 students

Late Stage Cell Culture Intern at Genentech – South San Francisco, CA May – August 2015

Tested enzyme inhibitors and activators as cell culture supplements to assess the impact on a specific

post-translational modification, PTM, for CHO cell lines in small scale and 2L bioreactors

Conducted 2L bioreactor runs to determine if PTM levels were dependent on clone or process parameters

Performed quantitative Western blots to determine differences in enzyme expression levels between

various cell lines and reactor runs

Collaborated with R&D department to explore CHO proteomics in search of specific enzymes

Developed leadership and presentation skills at a world-class pharmaceutical company

Research Fellowship in Biomolecular Engineering – University Park, PA May – August 2014

Performed molecular biology procedures such as restriction digest cloning and Gibson assembly

Utilized polymerase chain reaction and electrophoresis machines

Developed a biological control mechanism for trickle bed reactors used for plant propagation

Designed and constructed a vector to increase efficiency and cost effectiveness of plant transformation

Collaborated with graduate students and a visiting professor on projects involving bioreactor design and

recombinant protein expression

Perennials Supervisor, County Line Landscape Nursery – Harleysville, PA May – August 2013

Used extensive botanical knowledge to advise customers on purchases

Gained insight about the commercial ornamental herb and tree industries

PUBLICATIONS

Florez, S. L., Curtis, M. S., Shaw, S. E., Hamaker, N. K, Larsen, J. S., Curtis, W. R. A Temporary

Immersion Plant Propagation Bioreactor with Decoupled Gas and Liquid Flows. Biotechnology Progress

(Accepted December 15, 2015).

PRESENTATIONS

Nate Hamaker, Katie Legenski*, H Xu, W Muzika, Justin Yoo, Wayne Curtis (2015). Discovery,

Genome Assembly, and Interpretation of a Hydrocarbon-Producing Algae Symbiont. 2015 AIChE

Annual Conference, Nov. 8-13. Salt Lake City, UT.

Katie Legenski*, Nate Hamaker*, Justin Yoo, Wayne Curtis. (2015). Optical Mapping and NGS to

Characterize an Algae Biofuels Bacterial Symbiont. 2015 Bioinformatics and Genomics Retreat, Aug. 28-

29. University Park, PA.

*Designates pr esenter(s)

ACTIVITIES

Winner of AG Springboard Entrepreneurship Competition 2015

Grader for Chemical Engineering Fluid Mechanics (Ch E 330) 2015

Attended Conference as a Member of the Society for In Vitro Biology 2014

Mentor for Science-U Day Camp 2013

Member of Springfield Thon 2012 – 2013

HONORS, AWARDS, AND SCHOLARSHIPS

Schreyer Honors College Academic Excellence Scholarship Edward C. Hammond Jr. Memorial Scholarship

Leighton Riess Scholarship in Chemical Engineering Genentech Outstanding Student Award

Chemical Engineering Undergraduate Research Scholarship College of Engineering Dean’s List

Eberly College of Science Dean’s List James J. Kerrigan Memorial Scholarship