Analysis of Putative Sago Palm Cysteine Protease cDNA Open Reading Frame
JONG THIN THIN
Bachelor of Science with Honours
(Resource Biotechnology)
2013
Faculty of Resource Science and Technology
Analysis of Putative Sago Palm Cysteine Protease cDNA Open Reading Frame
Jong Thin Thin (26533)
A final project report submitted in partial fulfilment of the Final Year Project II
(STF 3015) Course
Supervisor: Associate Professor Dr. Hairul Azman @ Amir Hamzah Bin Roslan
Co-supervisor: Ms. Norafila Humrawali
Resource Biotechnology
Department of Molecular Biology
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
2013
II
DECLARATION
I declare that the thesis entitled “Analysis of Putative Sago Palm Cysteine Protease cDNA
Open Reading Frame” hereby submitted for the STF 3015 Final Year Project 2 at the
University Malaysia Sarawak (UNIMAS) is my own work and have not been previously
submitted by me at another University for any degree. I cede copyright of the thesis in
favor of the University Malaysia Sarawak (UNIMAS). Formulations and ideas taken from
other sources are cited as such. This work has not been published.
___________________
Jong Thin Thin (26533)
Biotechnology Resource
Faculty of Resource Science and Technology
University Malaysia Sarawak
I
ACKNOWLEDGEMENT
Upon completion of my final year project, I am contented and enjoyed the process of doing
the project. I did gain a lot of informative and technical knowledge by doing this
meaningful academic research study. Apart from the efforts of myself, the success of this
project depends largely on the encouragement and guidelines of many others. This research
project would not have been possible without the support of many of them. Therefore, I
take this opportunity to express my deepest gratitude for those who have been instrumental
in the successful completion of this project.
First and foremost, I would like to offer my special thanks to my supervisor, Associate
Professor Dr. Hairul Azman @ Amir Hamzah Roslan, for giving me an opportunity to
work on this project under his guidance. His valuable and constructive suggestions during
the planning and development of this research work are very vital for the success of this
project. His willingness to give his time so generously has been very much appreciated. I
also want to deliver special thanks to Ms. Norafila Humrawali for her guidance.
Besides that, I would also like to acknowledge the help provided by the master students of
Dr. Hairul, for the guidance, and sharing experiences and knowledge with me throughout
my project. Special thanks are also extended to my coursemates for sharing the literature
and invaluable assistance.
Finally, I wish to express my love and gratitude to my beloved families; for their
understanding, support, encouragement, and endless love, through the duration of my
studies.
III
TABLE OF CONTENTS
ACKNOWLEDGEMENT ........................................................................................................ I
DECLARATION ...................................................................................................................... II
TABLE OF CONTENTS ....................................................................................................... III
LIST OF ABBREVIATIONS .................................................................................................. V
LIST OF TABLES .................................................................................................................. VI
LIST OF FIGURES ............................................................................................................... VII
ABSTRACT ............................................................................................................................ IX
1.0 INTRODUCTION ............................................................................................................... 1
2.0 LITERATURE REVIEW ................................................................................................... 3
2.1 Cysteine Protease ......................................................................................................... 3
2.2 Cloning Vector ............................................................................................................. 5
2.2.1 The pET-41a(+) Expression System ..................................................................... 6
2.3 Restriction Enzyme ...................................................................................................... 8
2.4 DNA Ligase ............................................................................................................... 10
2.5 Bacterial Transformation Process in Plant ................................................................. 11
3.0 Materials and Method ....................................................................................................... 14
3.1 Media Preparation: Luria Broth (LB), Luria Agar (LA) and Calcium Chloride
(CaCl2) ............................................................................................................................. 14
3.2 Transformation of Escherichia. coli XL1 Blue with pBluescript Plasmid ................ 14
3.3 Isolation of Double-stranded pBluescript Plasmid DNA from E. coli XL1 Blue ..... 16
3.4 cDNA Insert Preparation............................................................................................ 17
3.5 The pET-41a(+) Vector Preparation .......................................................................... 19
3.6 DNA Extraction from Agarose Gel ........................................................................... 19
3.7 Restriction Endonuclease Digestion of DNA ............................................................ 20
3.8 DNA Ligation Process ............................................................................................... 21
3.9 Transformation of E. coli XL1 Blue with pET-41a(+)-msCPR cDNA ORF ............ 21
3.10 Screening Clone ....................................................................................................... 22
4.0 RESULTS ........................................................................................................................... 24
4.1 Transformation of E. coli XL1 Blue with pBluescript Plasmid which Containing
msCPR cDNA ORF ......................................................................................................... 24
IV
4.2 Isolation of Double-stranded pBluescript Plasmid DNA from E. coli XL1 Blue ..... 25
4.3 cDNA Insert Preparation............................................................................................ 26
4.4 The pET-41a(+) vector preparation ........................................................................... 27
4.5 Retriction digestion of DNA ...................................................................................... 28
4.6 Transformation of E. coli XL1 Blue with pET-41a(+)-msCPR cDNA ORF ............ 29
4.7 Screening Clone ......................................................................................................... 30
4.7.1 Colony PCR......................................................................................................... 30
4.7.2 Restriction Fragment Analysis ............................................................................ 31
4.7.3 DNA Sequencing ................................................................................................. 32
5.0 DISCUSSION ..................................................................................................................... 33
6.0 CONCLUSION .................................................................................................................. 43
7.0 REFERENCES .................................................................................................................. 44
V
LIST OF ABBREVIATIONS
msCPR Metroxylon sagu cysteine protease
DNA Deoxyribonucleic acid
cDNA Complementary deoxyribonucleic acid
IPTG Isopropyl-b-d-thiogalactopyranoside
LB Luria broth
LA Luria agar
CaCl2 Calcium chloride
NaCl Sodium chloride
Rpm Revolution per minute
Rcf Relative centrifugal force
OD 600 Optical density 600
°C Degree celcius
ml milliliter
Mm millimolar
% Percent
l Microliter
g Gram
PCR Polymerase chain reaction
EtBr Ethidium bromide
kb Kilobase pair
bps Base pairs
VI
LIST OF TABLES
Table 1: Recipe of master mix. ................................................................................................. 17
Table 2: The thermal cycler condition of the PCR process. ..................................................... 18
Table 3: Reagents needed for restriction endonuclease digestion of DNA. ............................. 20
Table 4: Reagents needed for DNA ligation process. ............................................................... 21
Table 5: Reagents needed for restriction endonuclease digestion of DNA. ............................. 22
VII
LIST OF FIGURES
Figure 1: Action of catalysis mechanism. ................................................................................... 3
Figure 2: The map of pET-41a(+) vector. .................................................................................. 7
Figure 3: The catalytic action of DNA ligase. (Adapted from: Sreedhara et al., 2004). .......... 10
Figure 4: Plant transformation. ................................................................................................. 12
Figure 5: Schematic diagram of the micro-shock wave devices. ............................................. 13
Figure 6: An agar plate that grown with colonies of E. coli XL1 Blue containing
pBluescript-msCPR cDNA ORF. .............................................................................................. 24
Figure 7: The photographic image of an agarose gel electrophoresis analysis of
pBluescript plasmid containing msCPR cDNA ORF which extracted from E. coli XL1
Blue. ........................................................................................................................................... 25
Figure 8: The photographic image of an agarose gel electrophoresis analysis of PCR
product of pBluescript plasmid which extracted from E. coli XL1Blue. .................................. 26
Figure 9: The photographic image of an agarose gel electrophoresis image of pET-41a(+)
plasmid....................................................................................................................................... 27
Figure 10: The photographic image of an agarose gel electrophoresis image of pET-41a(+)
plasmid and msCPR cDNA ORF that had been linearized with restriction enzyme, Nde I
and Not I. ................................................................................................................................... 28
Figure 11: An agar plate that grown with colonies of E. coli XL1 Blue containing pET-
41a(+)-msCPR cDNA ORF. ...................................................................................................... 29
Figure 12: The photographic image of an agarose gel electrophoresis analysis of PCR
product of pET-41a(+)-msCPR cDNA ORF plasmid which extracted from E. coli XL1Blue.
................................................................................................................................................... 30
Figure 13: The photographic image of an agarose gel electrophoresis analysis of the
comparison of pET-41a(+)-msCPR cDNA ORF plasmid that had been linearized with
VIII
restriction enzyme, Nde I and Not I with the uncut pET-41a(+) plasmid and pET-41a(+)-
msCPR cDNA ORF plasmid. .................................................................................................... 31
Figure 14: The nicked-open circular and supercoiled plasmid DNA. (Adopted from
VALUE, 2013). ......................................................................................................................... 37
Figure 15: The part of chromatogram of the data. .................................................................... 41
Figure 16: The part of chromatographic fluorescence data. ..................................................... 41
IX
Analysis of Putative Sago Palm Cysteine Protease cDNA Open Reading Frame
JONG THIN THIN
Resource Biotechnology
Department of Molecular Biology
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
Cysteine protease is one of the types of proteolytic enzyme that present in Metroxylon sagu and has many
roles in plant cells physiology and development process. In this study, the objective is to clone a putative
cysteine protease gene derived from M. sagu into an expression vector. Firstly, the msCPR cDNA ORF
which has been isolated from sago palm, was propagated in pBluescript plasmid. The clone was analyzed via
restriction enzyme and PCR. The second objective is to clone the msCPR cDNA ORF into an expression
vector, pET-41a(+). The last objective is to determine the correct insertion of the coding fragment of pET-
41a(+) which containing msCPR cDNA ORF through DNA sequencing reaction. A pBluescript vector
containing the putative msCPR was checked via PCR and produced a fragment with the size of 750 bps. Then,
the pET-41a(+) vector and msCPR cDNA ORF was successfully digested with Nde I and Not I restriction
enzyme and subsequently ligated together. However, the determination of the correct insertion of the coding
fragment of pET-41a(+)-msCPR cDNA ORF through DNA sequencing reaction was failed to be conducted.
Key words: Cysteine protease, pET-41a(+), restriction enzyme, ligation, DNA sequencing
ABSTRAK
Protease Cysteine adalah salah satu jenis enzim proteolitik yang terdapat dalam Metroxylon sagu yang
berperanan dalam sel-sel tumbuhan fisiologi dan proses pembangunan. Dalam kajian ini, objektif adalah
untuk mengklonkan putatif gen cysteine protease yang diperolehi daripada M. sagu ke dalam ekspresi vektor.
Pertama kali, msCPR cDNA ORF yang telah diasingkan daripada sagu telah dicantumkan dalam plasmid
pBluescript. Klon telah dianalisasikan melalui enzim retriksi dan PCR. Objektif kedua adalah untuk
mengklonkan msCPR cDNA ORF ke dalam vektor ekspresi, pET-41a(+). Objektif terakhir adalah untuk
menentukan kemasukan serpihan kod pET-41a(+) yang mengandungi msCPR cDNA ORF yang betul melalui
penjujukan DNA tindak balas. Vektor pBluescript yang mengandungi putatif msCPR telah diperiksa melalui
PCR dan menghasilkan serpihan yang bersaiz 750 bps. Kemudian, pET-41a(+) vektor dan msCPR cDNA
ORF telah berjaya dipotong dengan enzim retriksi Nde I dan Not I dan sterusnya pemasangannya bersama-
sama. Tetapi, pengenalpastian serpihan kod pET-41a(+)-msCPR cDNA ORF yang betul melalui penjujukan
DNA tindak balas telah gagal dijalankan.
Kata kunci: Protease cysteine, pET-41a(+), enzim retriksi, ligasi, penjujukan DNA
1
1.0 INTRODUCTION
Cysteine protease is one of the types of proteolytic enzyme that present in most of the
organisms which include animals, plants, some bacteria and viruses (Joo et al., 2007).
Cysteine protease carries out its function via catalytic mechanism whereby mediated
cleavage of a peptide bonds of a polypeptide. In plants, cysteine protease gene was shown
to be expressed during growth and development stage (Matarasso et al., 2006). Besides
that, Solomon and his colleagues (1999) investigated that cysteine protease plays an
essential role in the regulation of plant senescence and in the induction of programmed cell
death under stressed condition. Therefore, this enzyme also serves as a messenger in the
signaling pathway in order to response to the biotic and abiotic stress (Grudkowska &
Zagdariska, 2004). In addition, Mohan et al. (2008) had reported that cysteine protease
function as a defense protein by possessing a remarkable toxicity against insects and also
pathogens.
Cysteine protease comprise of more than 40 families which are further sub-
classified into at least six superfamilies (Grudkowska & Zagdariska, 2004). Grudkowska
and Zagdariska (2004) stated that most of the cysteine proteases that present in plant are
papain and legumain families. According to Peng et al. (2008), papain from the latex of
Carica papaya is the most widely studied cysteine protease in plant. This gene can also be
found in sago palm (Metroxylon sagu). Through the analysis of EST, Wee and Hairul
(2011) had found that cysteine protease in M. sagu is expressed when the plants encounter
stresses, such as pathogen attack. M. sagu is a very versatile and physically hard
monocotyledonous plant (Wee & Hairul, 2011). It is economically important and now
grown commercially in Malaysia for the starch production and also ethanol fuel production
as well as in food industry (McClatchey et al., 2006). Recently, there is an occurrence of
new competition in the production of biofuel and food due to the exhaustion of fossil
2
energy and the increment of world population (Ehara, 2012). M. sagu becomes a new
source needed to solve this problem because of its potential in the starch production (Ehara,
2012). In addition, it can tolerate salinity, prolonged flooding, and acidic peat soils
(McClatchey et al., 2006). Due to its economic value and adaptability towards such
environment, cysteine protease gene is isolated and the cloned into an expression system
for this study.
The discovery of isolation the msCPR cDNA ORF from sago palm prompted the
work to express the cysteine protease gene. Therefore, a vector containing msCPR cDNA
ORF was be constructed. The pET-41a(+) was selected in order to clone the msCPR cDNA
ORF because it has high level expression of recombinant protein and very suitable for
cloning. Before the cloning process, restriction enzyme was used to digest the msCPR
cDNA ORF and pET-41a(+) vector in order to produce sticky end which is complementary
with each other. Then, the positive clones were identified and followed by the
transformation process. DNA sequencing was conducted in order to ensure the correct
insertion of the coding fragment of pET-41a(+)-msCPR cDNA ORF.
In this study, there are three objectives to be taken into account. The first objective
is to obtain the msCPR cDNA ORF which has been isolated from sago palm from a
selected vector, called pBluescript. The second objective is to clone the msCPR cDNA
ORF which has been isolated from sago palm into a selected expression vector, pET-
41a(+). The last objective of this study is to determine the correct insertion of the coding
fragment of pET-41a(+) which containing msCPR cDNA ORF through DNA sequencing
reaction.
3
2.0 LITERATURE REVIEW
2.1 Cysteine Protease
Cysteine protease is a group of intracellular protease enzyme that plays a key role to
catalyse the cleavage of peptide bonds in a protein. This enzyme cleaves the bonds
between the amino acids in a polypeptide chain by hydrolysis. According to Grudkowska
and Zagdariska (2004), the protease enzymes can be classified into two types:
endopeptidase and exopeptidase. Endopeptidase cleave the peptide bonds on the interior
part of the polypeptide chain which is based on their active side residue; while,
exopeptidase cleave on the end part of the polypeptide chain which is based on their
substrate specificity (Grudkowska & Zagdariska, 2004). According to protease
classification, cysteine protease is an endopeptidase with a cysteine residue in their active
site. The action of catalysis mechanism is shown as follows:
Figure 1: Action of catalysis mechanism.
Cysteine protease consists of more than 40 families and sub-classified into at least
six clans (Grudkowska & Zagdariska, 2004). Among the protease enzyme, cysteine
protease is the first enzyme studied; and Schaller (2004) believed that this enzyme plays an
important role in plants. Papain is the first cysteine protease that was discovered in the
latex and fruit of Carica papaya, and the most extensively studied protease (Schaller, 2004).
4
Grudkowska and Zagdariska (2004) stated that the genome of Arabidopsis thaliana
contains the gene which codes for 32 types of papain-like cysteine protease.
From the previous study, they found out that among 42 proteinases, there were 27
cysteine proteases took part in barley seed germination. In maize, 90% of the degradation
activity was controlled by cysteine protease (Grudkowska & Zagdariska, 2004). Therefore,
this indicates that cysteine protease plays a vital role in protein degradation and
mobilization. Besides that, Solomon (1999) stated that programmed cell death is occurred
as the cysteine protease is being induced by environmental-stress. Grudkowska and his
coworkers (2004) had proved that wheat can tolerate frost conditions because of the
activity of cysteine protease gene in their genome. In addition, the plant cysteine proteases
also serve as a defense mechanism by possessing toxicity against insect larvae such as for
papain from papaya (Carica papaya) that has been proved to act in the defense mechanism
against insect larvae.
A study conducted by Nieuwenhuizen et al. (2012) stipulated that cysteine protease
in the kiwifruit has an ability in the application of degradation of gelatin. Cysteine protease
enzyme also plays an important role in the medical field to treat allergenicity
(Nieuwenhuizen et al., 2012). This indicates the great functions of the cysteine protease in
the plant.
5
2.2 Cloning Vector
Vector is known as a small piece of DNA molecule which serves as an agent to carry a
gene from the donor to the host cell. In the era of biotechnology, vector is widely used for
DNA cloning in order to produce genetically modified organisms. As stated by Levine
(2007), cloning was first invented at the twentieth century and this technology was
successfully created a cloned sheep in 1996. This cloned sheep project had prompted the
more advanced work of producing transgenic organisms. On the other hand, Goldberg
(2001) stipulated that plant cloning era was established at the end of the 1970s after the
principles of plant genome organization and gene regulation were known by the scientists.
John Bedbrook and his colleagues had proved that plant DNA can be cloned and replicated
in bacteria as other organisms DNA (Goldberg, 2001). They had successfully cloned the
ribosomal DNA and telomeric repeated sequences from wheat into the vector system by
using the same enzymes which are similar to all other organisms used (Goldberg, 2001).
After that, many plant genome libraries had been constructed in the early 1980s by
using the vector cloning method. Those constructed libraries make the plant genome
available to scientists all over the world. Besides that, Goldberg (2001) had mentioned that
plant cDNA libraries were also constructed in order to study the plant gene expression at
the level of transcription. At the same time, developmental, metabolic, as well as
biochemical process of plant can be studied through the discoveries of plant cDNAs.
There are many researches such as the establishment of Arabidopsis and rice
genome projects had been successfully discovered by using this technology (Goldberg,
2001). Many sequencing projects which had helped to uncover tens of thousands of mRNA
in a wide range of plants by using the technology of expressed sequence tag (Goldberg,
2001). In addition, many transgenic plants have been created with invention of the vector
cloning technique. In 2000, golden rice had produced by modified the metabolic pathway
6
and subsequently introduced into the endosperm of rice, so that high yield of beta carotene
can be obtained and vitamin A can be produced in the large quantities (Key et al., n.d.).
In the modern plant biology and biotechnology era, many new discoveries and
robust development have been introduced by the scientists. New vector system has been
introduced in order to facilitate the simple cloning strategies and also high efficiency in the
gene expression. A set of ligation-independent cloning vectors has been launched for the
functional studies in plants (Rybel et al., 2011). This type of vector is very effective
because moderately high throughput fashion can be applied in verifying protein
localizations. Precise cloning is allowed by using ligation-independent cloning vectors
(Rybel et al., 2011). The advancement in this field prompted the work in the field of plant
molecular study.
2.2.1 The pET-41a(+) Expression System
The pET expression system is widely used in the field of cloning or recombinant DNA due
to its high efficiency in transcription and translation (Agilent Technologies, 2012). Protein
expression of pET system can be induced by using IPTG. The pET expression vectors are
originally derived from pBR322 plasmid, are engineered in order to establish a system with
high efficiency in transcription and also translation (Agilent Technologies, 2012). For the
pET-41 series, it is specifically designed for the high level of protein expression through
the popular GST fusion tag (Novagen, n.d.).
7
Figure 2: The map of pET-41a(+) vector.
. (Adapted from: Novagen, n.d.).
8
2.3 Restriction Enzyme
Restriction enzyme is an enzyme which functions to cleave the DNA at the recognized
specific site; often four, six or eight base pairs long, and subsequently to produce
fragments. The discovery of restriction enzyme is a great contribution in the field of
biotechnology as it serves as an important tool in the DNA recombinant technology
toolbox and makes genetic engineering possible. According to Nwankwo and Abalaka
(2011), restriction enzyme is found in the bacteria and some archaea bacteria which serve
as a defense mechanism to against invading viruses.
According to Nwankwo and Abalaka (2011), restriction enzyme was discovered by
a Swiss microbiologist, Werner Arber who received 1978 Nobel Prize in Physiology and
Medicine. Werner Arber and his colleagues found out that bacterial cell able to self-
defense against the foreign DNA through the study of phenomenon of host-controlled
restriction of bacteriophages (Pray, 2008). During that study, they observed that phage
particles are often unable to grow well and infect other strains of the same bacterial species
as they can grow well and efficiently infect one strain of bacteria (Pray, 2008). Besides that,
phage particles show opposite pattern which are able to grow well and infect a second
strain of bacterial species efficiently as they grow poorly in the original strain (Pray, 2008).
Furthermore, the study also showed that phage particles which able to grow and infect host
cells efficiently possess a DNA modification by the addition of methyl groups to the
adenine or cysteine bases, while the phage particles which only show poorly infection to
their host cells that does not have DNA methylation pattern (Pray, 2008). Pray (2008)
highlighted that phage particles were said to be restricted by their host as they only grow
and infect poorly to their host cells.
There are different types of restriction enzymes have been created. Restriction
enzymes are categorized into four categories, which are Type I, Type II, Type III, and also
9
artificial restriction enzymes (Nwankwo & Abalaka, 2011). Different restriction enzymes
cleave at different sequence of nucleotides and produce double-stranded cut in the DNA
which are differ in sequence, length, and strand orientation of a sticky end and also blunt
end (Nwankwo & Abalaka, 2011). As postulated by Pray (2008), “Type I restriction
enzyme recognize specific DNA sequences but cut at the random sites that can be as far as
1000 base pairs away from the recognition site; Type II recognize and cut directly within
the recognition site; and Type III recognize specific sequences but cut at different specific
location that is usually within about 25 base pairs of the recognition site”. Whereas,
artificial restriction enzymes are created by fusing engineered DNA binding domain to a
nuclease domain; so that, it is able to bind to desired DNA sequences (Nwankwo &
Abalaka, 2011). The most commonly used artificial restriction enzymes especially used in
genetic engineering applications and also standard gene cloning applications are zinc finger
nucleases (Nwankwo & Abalaka, 2011).
Nowadays, restriction enzymes play an important in genetic engineering as it helps
to insert the desired genes that have been cut into the plasmid vectors for gene cloning
purposes (Nwankwo & Abalaka, 2011). Besides that, it also widely used in polymerization
chain reaction (PCR) DNA-base manipulation in-vitro, molecular husbandry, southern
blotting analysis genetic engineering, restriction fragment length polymorphism (RFLP)
analysis, DNA mapping, and many others application in biotechnology (Nwankwo &
Abalaka, 2011).
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2.4 DNA Ligase
DNA ligase is a kind of enzyme that widely used in catalyzing the formation of
phosphodiester bond to join the 5’ phosphate of one strand of DNA to the 3’ hydroxyl of
another strand of DNA. ATP, as the energy source is required for this catalytic reaction to
carry out efficiently. According to Ellenberger and Tomkinson (2008), DNA ligases play a
vital role in DNA replication, repair, and also recombination. The catalytic action of DNA
ligase is illustrated in Figure 3.
Figure 3: The catalytic action of DNA ligase. (Adapted from: Sreedhara et al., 2004).
DNA ligase has become an important tool in the molecular biology research for
genetic engineering purposes. It helps to join the desired gene fragment into the selected
plasmid vector in order to produce a genetically modified gene products. T4 DNA ligase is
the most widely used DNA ligase in the field of molecular biology. T4 DNA ligase is a
single polypeptide which derived from T4 bacteriophage (VALUE, 2013).
11
2.5 Bacterial Transformation Process in Plant
Transformation is processes that only can be carried out by bacteria to uptake free DNA
actively and subsequently integrate the genetic information into the cell (Lorenz &
Wackernagel, 1994). As highlighted by Lorenz and Wackernagel (1994), transformation
was first invented by Griffith who had discovered the transformation in Streptococcus
pneumonia in 1928. Avery and his coworkers had demonstrated that “DNA is a
transforming principle and came out some ideas about bacteria might be favored subjects
for genetic investigation and eventually for technological application of molecular genetics
science” (Lederberg, 1994).
Through this idea, bacterial transformation had become very popular in many
studies in the past. The bacteria have been widely investigated include Pneumococcus,
Haemophilus influenza, Bacillus subtilis, and some others bacteria (Goldberg, 2001).
Previously, some scientists thought that Escherichia coli were not responding to
transformation. However, Cohen and his coworkers (1972) had claim that E. coli can be
transformed with the treatment of calcium chloride. The discovery of E. coli
transformation had prompted the work of discovery of artificially-induced competence in E.
coli which is simpler to carry out for molecular cloning (Cohen et al., 1972). Froger and
Hall (2007) had reported that transformation of plasmid DNA into E. coli can be done by
using the heat shock method. Besides that, Singh et al. (2010) found out that
transformation efficiency was ~24 fold higher when the calcium chloride treated cells were
further incubated on ice for 10 min after heat shock when compared to no heat shock and
only heat shock treatment. Transformation is now extensively used in molecular biology
field in order to produce transgenic organism. The illustration of plant transformation is as
follows:
12
Figure 4: Plant transformation.
(Adapted from: Genetically Engineered Crops, n.d.).
Furthermore, many researchers had started focus on the study of Agrobacterium
tumefaciens and its causative agent at around 90 years ago (Riva et. al., 1998). They found
out that A. tumefaciens has an ability to integrate its DNA of the tumor-inducing plasmid
into the nucleus of the host cells. Dohbal et al. (2010) used neomycin phosphotransferase
II (npt II) and glucuronidase (GUS) as reporter genes in order to study the transformation
efficiency of using A. tumefaciens. According to Dohbal et al. (2010), A. tumefaciens is the
most used method for the plant transformation process whereby help in delivery of gene of
interest into a host nuclear genome with the high transformation frequency.
Recently, there is a new invention for bacterial transformation, which is carried out
in the more efficient and cheaper way. Prakash and his colleagues (2011) generated a
unique device, which is a 30 centimeters long explosive coated polymer tube to create a
micro-shock wave that facilitate the entry of DNA from the surrounding into the cells of
bacteria. Prakash et al. (2011) believed that the momentum disturbance that resulted from
the shock wave can change the bacterial cell membrane permeability to facilitate the
uptake of the DNA from the surrounding. According to Parkash et al. (2011), the
efficiency of transformation by using this method is higher as electroporation technique,
13
and it has a benefit of better recovery of cells, and it is rather cheaper than the commercial
electroporation.
Figure 5: Schematic diagram of the micro-shock wave devices.
(Adapted from: Prakash et al., 2011).
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