Engineering control of bacterial cellulose production using a genetic toolkit and

a new cellulose producing strain

Michael Florea1,2,3, Henrik Hagemann1,4, Gabriella Santosa1,2, James Abbott5,6, Chris N. Micklem1,2, Xenia Spencer-Milnes1,2, Laura

de Arroyo Garcia1,2, Despoina Paschou1,4, Christopher Lazenbatt1,2, Deze Kong1,4, Haroon Chughtai1,4, Kirsten Jensen1,7, Paul

Freemont1,7, Richard I Kitney1,4, Benjamin Reeve1,4, Tom Ellis1,4

Author affiliation: 1Centre for Synthetic Biology and Innovation, Imperial College London, London SW7 2AZ, UK. 2Department of

Life Sciences, Imperial College London, London SW7 2AZ, UK. 3Department of Biosystems Science and Engineering, ETH Zürich,

Mattenstrasse 26, 4058 Basel, Switzerland. 4Department of Bioengineering, Imperial College London, London SW7 2AZ, UK.

5Bioinformatics Support Service, Department of Surgery and Cancer, Imperial College London, London SW7 2AZ, UK. 6Centre for

Integrative Systems Biology and Bioinformatics, Imperial College London, London SW7 2AZ, UK. 7Department of Medicine,

Imperial College London, London SW7 2AZ, UK.

Corresponding author: Dr Tom Ellis, Centre for Synthetic Biology and Innovation, Imperial College London, London SW7 2AZ,

UK. Tel: +44 (0)20 7594 7615, Email: t.ellis@imperial.ac.uk

Keywords: synthetic biology, bacterial cellulose, genetic engineering, biomaterials, genomics

BIOLOGICAL SCIENCES: Microbiology

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ABSTRACT

Bacterial cellulose is a strong and ultrapure form of cellulose produced naturally by several species of the Acetobacteraceae.

Its high strength, purity and biocompatibility make it of great interest to materials science, however precise control of its

biosynthesis has remained a challenge for biotechnology. Here we isolate a new strain of Komagataeibacter rhaeticus

(Komagataeibacter rhaeticus iGEM) that can produce cellulose at high yields, grow in low nitrogen conditions, and is highly

resistant to toxic chemicals. We achieve external control over its bacterial cellulose production through development of a

modular genetic toolkit that enables rational reprogramming of the cell. To further its use as an organism for biotechnology,

we sequenced its genome and demonstrate genetic circuits that enable functionalization and patterning of heterologous

gene expression within the cellulose matrix. This work lays the foundations for using genetic engineering to produce

cellulose-based materials, with numerous applications in basic science, materials engineering and biotechnology.

SIGNIFICANCE STATEMENT

Bacterial cellulose is a remarkable material that is malleable, biocompatible and over 10 times stronger than plant-based

cellulose. It is currently used to create materials for tissue engineering, medicine, defence, electronics, acoustics and fabrics.

We describe here a new bacterial strain that is readily amenable to genetic engineering and produces high quantities of

bacterial cellulose in low-cost media. To reprogram this organism for biotechnology applications, we created a set of genetic

tools that enables biosynthesis of patterned cellulose, functionalization of the cellulose surface with proteins and tunable

control over cellulose production. This greatly expands our ability to control and engineer new cellulose based biomaterials,

offering numerous applications for basic research, materials science and biotechnology.

INTRODUCTION

The emergence of synthetic biology now enables model microorganisms such as Escherichia coli to be easily reprogrammed

with modular DNA code to perform a variety of new tasks for useful purposes (1). However for many application areas it is

instead preferable to exploit the natural abilities of non-model microbes as specialists at consuming or producing molecules or

thriving within niche environments (2). Recent work has described adapting common E. coli synthetic biology tools to work

across different bacterial phyla (3, 4) and has produced genetic toolkits for new bacteria, where collections of DNA constructs

and methods for precise control of heterologous gene expression have been developed for engineering strains naturally

specialised for photosynthesis or survival within the gut microbiome (5, 6). An important application area for biotechnology is

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the production of materials, and bacteria that naturally secrete high yields of cellulose have attracted significant attention not

just from people in industry and research (7), but also from those in art, fashion and citizen science (8). However, despite their

widespread use, no toolkit for genetic modification of these cellulose-producing bacteria has previously been described.

Komagataeibacter is a genus from the Acetobacteraceae family of which multiple species produce bacterial cellulose. Cellulose

nanofibers are synthesized from UDP-glucose by the acs (Acetobacter cellulose synthase) operon proteins AcsA and AcsB (9)

and secreted by AcsC and AcsD, forming an interconnected cellulose “pellicle” around cells (7). Although it is still unclear why

Acetobacteraceae produce bacterial cellulose in nature (7), it has been shown to confer the host a high resistance to UV light

and a competitive advantage in colonization over other microorganisms (10). In materials science, genetic engineering has been

used to create several novel biomaterials, such as strong underwater protein-based adhesives (11), self-assembling,

functionalized amyloid-based biofilms (12), biodegradable bacterial cellulose based tissue engineering scaffolds (13), and many

others. Bacterial cellulose has long been a focus of research because unlike plant-based cellulose, it is pure from other chemical

species (lignin and pectin) and is synthesized as a continuous highly interconnected lattice (14). This makes the material

mechanically strong (nanocellulose fibres possess tensile stiffness of between 100-160 GPa and tensile strength of at least 1

GPa (15, 16)), while still flexible, biocompatible and highly hydrophilic, capable of storing water over 90% of total weight (17,

18). Due to these properties, bacterial cellulose is commercially used in medical wound-dressings, high-end acoustics and many

other products (7), and in the laboratory, has been used to create biodegradable tissue scaffolds (13), nano-reinforcements

(19), artificial blood vessels (18), as well as sensors (20), flexible electrodes (21), OLED displays and other materials (see Hu et

al., 2014) (22).

Functionalization or modification of bacterial cellulose has mainly been achieved by chemical or mechanical modifications of

the cellulose matrix or via changing culturing conditions (7, 22), while only a few attempts at genetic engineering have been

made (13, 23). However, genetic engineering may allow a greater range of materials to be produced, by enabling fine control

over cellulose synthesis genes and production of protein-cellulose composite biomaterials. Here we isolate a new strain of

Komagataeibacter rhaeticus (previously Gluconaceteobacter rhaeticus) (24) that can grow in low nitrogen conditions and

produce cellulose at high yields, sequence its genome, and develop a synthetic biology toolkit for its genetic engineering. This

toolkit provides the characterization data necessary for engineering of K. rhaeticus iGEM, and enables transformation,

controlled expression of constitutive and inducible transgenes, and control over endogenous gene expression of this strain. We

use these tools to engineer a system that allows tunable control over native cellulose production, and produce novel patterned

and functionalized cellulose-based biomaterials.

RESULTS

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Isolation, characterization and genome sequencing of Komagataeibacter rhaeticus iGEM

As part of the International Genetically Engineered Machine Competition (iGEM) (25), we evaluated Gluconacetobacter

hansenii ATCC 53582 (one of the highest reported cellulose producing strains (26), recently reclassified as Komagataeibacter

hansenii ATCC 53582) (27), and a new strain isolated from Kombucha tea as potential new synthetic biology hosts (Fig. 1a). We

chose the latter strain (hereafter called ‘iGEM’) for further work, as preliminary experiments showed that it can be transformed

more readily with plasmid DNA than G. hansenii ATCC 53582. Furthermore, the iGEM strain produced more cellulose than G.

hansenii ATCC 53582 on sucrose in small-scale tests (Fig. 1b) and also produced cellulose at high yields on cheap, low-nitrogen

Kombucha tea medium (Fig. 1b). Surprisingly, it could also grow on the defined nitrogen-free LGI medium (Fig 1c, d,

Supplementary Fig. S1, S2). As several species of Acetobacteraceae, notably Gluconacetobacter diazotrophicus have been

confirmed to fix atmospheric nitrogen (28, 29), this suggested possible nitrogen fixation (see below). Finally, as cellulose has

been reported to increase resistance towards UV and environmental stresses in closely related species (10), we tested whether

cellulose could also confer resistance towards chemical stresses, which may occur in unrefined feedstocks or during industrial

production. We tested the susceptibility of the iGEM strain to 70% ethanol, 10% bleach, 0.1 M NaOH and 0.1 M HCl (Fig. 1e),

and found that when encased in cellulose it is highly tolerant to chemical stressors, being over 1000-fold more resistant than E.

coli to all treatments (Fig. 1e). Finally, scanning electron microscopy of a cellulose pellicle confirmed that as with other closely

related species, cells of K. rhaeticus iGEM are rod-like, approximately 2μm in length, and are heavily encased in cellulose during

normal growth (Fig. 1f, Supplementary Fig. S3).

To determine the genetic basis of high cellulose productivity and to provide background information for genetic engineering of

the iGEM strain, we sequenced its genome to 400x coverage, and assembled the genome using the genome of

Gluconacetobacter xylinus NBRC 3288 (31) as the reference genome for scaffolding (ENA project ID PRJEB10933). Sequencing

showed that genome totals 3.87 Mbp with a GC% of 62.7 and contains a predicted 3572 genes, with an N50 of 3.16 Mbp. The

genome is divided between a chromosome of 3.16 Mbp, at least two plasmids – pKRi01 (238 kbp) and pKRi02 (3 kbp), and 37

unplaced contigs (in total 460 kbp) which may be part of the chromosome or additional plasmids, and could not be confidently

assigned due to being flanked by repetitive sequences (Fig. 2a). The genome sequence revealed several interesting aspects

about the biology of K. rhaeticus iGEM. Firstly, a 16s rRNA phylogeny suggests the iGEM strain to be a new strain of

Komagataeibacter rhaeticus (Supplementary Fig. S4), rather than Gluconacetobacter xylinus which is normally thought to be

associated with Kombucha tea. Furthermore, the sequence shows the presence of 4 acs cellulose synthesis operons on the

chromosome, sharing 40-65% amino acid identity (Fig. 2a,b,c). Up to 3 acs operons have been reported in other bacterial

cellulose producing species (in G. xylinus ATCC 23769 and G. hansenii ATCC 53582 (32, 33)), indicating that the high cellulose

synthase copy number may be a possible contributor to the high cellulose productivity observed here (Fig. 1b). These operons

also differ in structure. The acs1 operon contains separate acsA and acsB genes, while they are fused in the other operons, and

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the only genomic copy of acsD is found in acs1. Operon acs4 uniquely contains only acsAB genes, and phylogenetic analysis

indicates that acs4 is most closely related to the acs2 operon (Fig. 2b), and possibly arose via duplication and subsequent

translocation. From the genes flanking acs operons, cmcAX, ccpAX, bglxA, bcsX and bcsY have been previously shown to

contribute to cellulose production in closely related species (26, 34, 35). We found two other, standalone copies of bglxA from

the genome (genomic position 517401 - 519440 and 3029825 - 3032221), and also identified genes close to acs2 that are

associated with extracellular matrix formation (kpsC, kpsS and rfaB) and may play a role in cellulose productivity (36, 37).

Finally, to determine whether the iGEM strain can fix atmospheric nitrogen similarly to G. diazotrophicus, we searched its

genome for genes associated with nitrogen fixation. We located 5 genes (ntrB,C,X,Y and nifU - Supplementary Table S1)

associated with nitrogen fixation in Acetobacteraceae (38), however interestingly we did not find the genes homologous to

nifHDK, which form the main nitrogenase subunits in G. diazotrophicus.

Genetic engineering toolkit for Komagataeibacter

Very few genetic tools, with little characterization data are available for engineering of Acetobacteraceae. We therefore

developed a complete set of tools for its engineering, consisting of protocols, modular plasmids, promoters, reporter proteins

and inducible constructs that enable external control of gene expression (Fig. 3a).

Protocols and plasmid backbones - We first used the plasmid pBla-Vhb-122 (previously described to replicate in

Acetobacteraceae) (39) to develop protocols for the preparation of electrocompetent cells, transformation, plasmid purification

and genomic DNA extraction of K. rhaeticus iGEM (see Supplementary Protocols). Using these protocols, we then assessed 8

plasmids for propagation in K. rhaeticus iGEM - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351; pBAV1K-T5-sfGFP,

pSB1C3 and pBca1020 (see Supplementary Table S2 for details). From these, pSEVA321, 331, 351; pBAV1K-T5-sfGFP and pBla-

Vhb-122 showed replication in iGEM (Supplementary Fig. S5) giving a total of 5 different plasmids to act as vectors . We further

engineered pSEVA321 and pSEVA331 into pSEVA321Bb and pSEVA331Bb, making them compatible with the widely-used

BioBrick standard (25), to enable rapid cloning of publically-available DNA parts. We then used pSEVA331Bb for all subsequent

studies, due to its likely higher copy number.

Reporter proteins, constitutive and inducible promoters - We next tested expression of 7 reporter proteins (mRFP1, GFPmut3,

sfGFP, and chromoproteins tsPurple, aeBlue, gfasPurple and spisPink; see Supplementary Table S3 and S4 for details), from

which mRFP1, GFPmut3 and sfGFP showed visually detectable expression. We then chose 10 promoters from an open-access

collection of synthetic minimal E. coli promoters and using mRFP1 as the reporter, characterized these in K. rhaeticus iGEM (Fig.

3b and Supplementary Table S4, also see Supplementary Fig. S6 for a comparison to promoter strengths in E. coli). For inducible

promoters, we engineered 4 constructs allowing gene expression to be induced externally by anhydrotetracycline (ATc) or N-

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acyl homoserine lactone (AHL) (see Supplementary Fig. S7 for overview of constructs). From these, the AHL-inducible constructs

(pLux01 and pLux02) showed higher induction and lower leakiness than the ATc-induced constructs (pTet01, pTet02) (Fig. 3c),

and contrary to our initial expectations, they also gave robust induction of mRFP1 expression when cells were encased in the

pellicle, showing visible fluorescence (Fig. 3d, e, Supplementary Fig. S8). This is notable as it shows that cells in the pellicle can

effectively receive signals from their environment despite their cellulose encasing. As K. rhaeticus is highly resistant to various

environmental hazards within cellulose (Fig. 1e), the ability to receive signals while protected by cellulose makes it a potentially

suitable host for applications requiring tolerance to toxic chemicals and long-term survival.

Engineering control over cellulose production

As wild-type species produce cellulose constitutively, a major goal of genetic engineering of Acetobacteraceae has been to

achieve control over cellulose production. Constitutive cellulose production complicates genetic engineering techniques and is

not always desirable for industrial applications as it is imparts a high metabolic cost, which in well-aerated conditions typically

leads to the emergence of cellulose-nonproducing mutants (40). It is therefore desirable to inhibit cellulose production during

periods when it is not required, in order to prevent the proliferation of these mutants. Furthermore, fine control over cellulose

production levels may allow control over the density of cellulose fibrils, and thus the macroscale properties of cellulose. To

achieve controlled cellulose production, we engineered a system in which an E. coli Hfq, and an sRNA targeting UGPase mRNA

(UDP-glucose pyrophosphorylase) are co-expressed from a plasmid in response to AHL (plasmid J-sRNA-331Bb; Fig. 4a, also see

Supplementary Fig. S9 and Supplementary File S1 for detailed overview). The sRNA contains a 24 base region complementary to

UGPase mRNA and an E. coli Hfq binding region. When expressed, it binds to the target UGPase mRNA and recruits E. coli Hfq,

inhibiting UGPase translation. We targeted the UGPase gene as it catalyses the production of UDP-glucose critical for cellulose

synthesis (41) and is present in single-copy in the genome, allowing knockdown by a single sRNA. We found this system to be

highly efficient, as cellulose production was suppressed completely upon full induction and could be fine-tuned using different

concentrations of AHL (Fig. 4b). The observed reduction in cellulose production was not related to toxicity, as growth rate did

not decrease compared to wild type levels (Fig. 4c, Supplementary Fig. S10). This system was engineered to be a general

platform for targeted knockdowns in Komagataeibacter and other bacterial species, as expression of E. coli Hfq makes it

independent from the host Hfq and the broad host range pSEVA331Bb backbone enables replication in a wide range of species.

Furthermore, new sRNAs can be added to the plasmid, and the 24 base sRNA region can be recoded rapidly by site-directed

mutagenesis, making the construct easily modifiable for other targets.

Genetic engineering of patterned and functionalized biomaterials

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Owing to the discovery that K. rhaeticus gene expression can be induced even when inside a cellulose pellicle, we hypothesized

that it may be possible to generate spatially and temporally patterned biomaterials that are controlled by the diffusion of the

inducer AHL and timing of exposure to induction during pellicle growth. To test this, we induced growing cellulose pellicles with

cells containing the AHL-inducible construct pLux01 with different concentrations of AHL, at different locations and time points

(Fig. 5a, b). We found that both spatial and temporal control were possible. When a limited amount of inducer was added to

one side of the pellicle, cells produced mRFP1 following the diffusion gradient of AHL (Fig. 5a). Furthermore, as cells are active

only in the top layer of the cellulose pellicle (7), when inducer was added at different times mid-way through pellicle growth,

only cells at the growing top layer produced mRFP1, capturing the temporal difference between uninduced cells in the bottom

layers and induced cells at the top (Fig. 5b).

To produce functionalized cellulose materials where the nanocellulose matrix is coated by proteins of interest, we considered

two strategies: genetic engineering of K. rhaeticus to produce these proteins in situ, or separate expression of proteins in E. coli

which are then purified and applied directly to bacterial cellulose (Fig. 5c). While the latter requires a three-step process

(protein and cellulose production separately, followed by combining the two), it may be preferred for medical and other

applications where very high purity of the material is required, as it would allow defined and purified components to be used

for functionalization. To test for the possibility of post-hoc functionalization, we produced mRFP1 in E. coli, extracted and added

it to bacterial cellulose, and compared it by fluorescence microscopy to cellulose produced by K. rhaeticus with in vivo

constitutive mRFP1 expression. We found that extracted proteins can diffuse well throughout the pellicle and functionalize the

cellulose evenly (Fig. 5d), while the granular fluorescence exhibited by expression from pellicle-based cells (see Fig. 3e, also

Supplementary Fig. S11 for full-size comparison) indicates that mRFP1 remains largely in the K. rhaeticus cells and would likely

require active secretion or lysis of cell membranes to access the extracellular cellulose.

To further increase efficiency of functionalization, we engineered expression vectors that allow easy fusion of proteins to one of

4 different cellulose binding domains (CBDs) – CBDclos (42), CBDCex (43), dCBD (44) and CBDcipA (45). CBDs are short peptides

that bind tightly to cellulose fibrils, thus increasing protein adhesion to cellulose (46). In these constructs, proteins can be

modularly fused to CBDs via restriction enzyme cloning. We assessed the cellulose binding strengths of these CBDs by washing

four different E. coli-extracted CBD-sfGFP fusion proteins with different solvents (dH20, EtOH, BSA and PBS) and measuring the

fluorescence that remained bound. Addition of CBDs to sfGFP gave up to a 5-fold increase in binding to cellulose when

compared to GFP alone (Supplementary Fig. S12). Finally, as bacterial cellulose is a candidate for new textile materials and of

high interest to the fashion industry, we used our approach to demonstrate production of functionalized garments. Using the

CBDcipA-sfGFP fusion protein extract and dried pellicle material from K. rhaeticus cultures, we created bacterial cellulose

fashion accessories by functionalization of cellulose fibrils with green fluorescent protein (Supplementary Fig. S13), indicating

that this approach is scalable to produce macroscale objects.

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DISCUSSION

Here we isolated a new strain of bacterial cellulose producing K. rhaeticus (K. rhaeticus iGEM), which can grow on acidic and

low-nitrogen media, is highly resistant to damaging chemical agents when encased in cellulose, and is notable for its high

cellulose production. Although it is clear that K. rhaeticus iGEM can effectively produce cellulose in low-nitrogen media,

whether it fixes nitrogen is still an open question. Nitrogen fixation seems to be prevalent in Acetobacteraceae, as multiple

species (most notably Gluconacetobacter diazotrophicus) have been recorded to be nitrogen fixing (47). However, in the case of

K. rhaeticus iGEM, although it can grow in nitrogen-free LGI medium (Fig. 1c, d; Supplementary Fig. S1, S2) and its genome

contains a set of genes associated with nitrogen fixation (Supplementary Table S1), we were unable able to find genes

homologous to G. diazotrophicus nifHDK, which form the structural subunits of the nitrogenase complex (48). This either

suggests that despite careful handling, very low level nitrogen contamination may have been present, allowing K. rhaeticus to

produce cellulose in LGI medium, or alternatively that K. rhaeticus uses a different set of nitrogenase genes for nitrogen

fixation, as alternative nitrogenases have been isolated in other species (49). However in either case, from the perspective of

manufacturing, low nitrogen tolerance allows the use of cheap, low-nitrogen media, potentially reducing cellulose

manufacturing costs.

To use this strain for biotechnology applications, it was first necessary to develop methods and tools for its genetic

manipulation. For a genetic toolkit to be useful, it should minimally allow introduction of foreign DNA, plasmid propagation and

a degree of control over heterologous gene expression levels. Although the required tools can differ between species and the

applications they are used in, they should ideally allow tunable regulation of transgene expression through inducible or

repressible systems and regulation over native gene expression. They should further provide relevant characterization data

about the host, such as a genome sequence, and growth and productivity rates in different conditions. Indeed, many of these

elements have been part of toolkits created for other species (5, 6, 50). The toolkit described here for K. rhaeticus iGEM

contains all of these features, providing protocols and DNA constructs to allow transformation, precisely controlled constitutive

and inducible expression of heterologous genes, control over endogenous gene expression, and a genome sequence with

characterization data.

Using this toolkit, we created two approaches for engineering new cellulose-based materials - first through genetic engineering

of K. rhaeticus itself, and second through application of extracted proteins to the bacterial cellulose. Genetic engineering offers

simple, one-step in situ production of materials and would be ideal for applications where the cellulose matrix is modified as it

is being made. The physical and biochemical properties of cellulose are largely dependent on the microscale morphology and

structure of cellulose fibrils, so modification of these during production by expressed heterologous enzymes could result in new

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material properties. For example, by engineering cells to incorporate N-acetylglucosamine residues into cellulose, Yadav et al.

(2010) (13) created biodegradable, low immunogenicity cellulose tissue scaffolds. Our second approach (addition of purified

proteins to already-produced cellulose) is likely to be preferable for applications where bulk material properties are not

changed, but the material is functionalised with new properties. In our experiments, post-hoc functionalization resulted in an

even, thorough distribution of functionalizing proteins (Fig. 5d), and may be preferable in medical and tissue engineering

applications where highly-pure materials are required, as both the cellulose and functionalizing proteins can be purified prior to

formation of the composite material. Together these two approaches complement each other, as the ability to modify cellulose

via genetic engineering as well as functionalize it with purified proteins allows for a wider range of materials to be engineered.

Here, genetic engineering allowed us to control cellulose production by sRNA-mediated knockdown, and create spatial and

temporal patterning through induction with N-acyl homoserine lactone (AHL). We used an AHL-inducible system here, however

for industrial applications it is likely straightforward to exchange it for a control system using cheaper chemical or physical

inducers if required. While we did not test for this specifically, it is important to note that as knockdown of cellulose production

did not significantly change growth rate, control over cellulose production levels may also allow control of the specific cell-vs-

cellulose and thus protein-vs-cellulose concentrations in the material. In the case of functionalization of cellulose with CBD-

fusion proteins, in principle any protein stable enough to function in the intended extracellular environment could be used for

functionalization.

By providing genetic engineering tools that allow control of production of bacterial cellulose and modification of it as a

material, this work enables a variety of potential applications. The physical properties and pore size of the cellulose might be

tunable by altering gene expression of the K. rhaeticus acs operons or by complexing cellulose in situ with secreted structural

proteins such as curli fimbriae (12). Cellulose can be functionalized with specific proteins (see Fig. 5c, Supplementary Fig. S12,

S13), which may be used to create advanced materials such as bacterial cellulose wound-dressings coated with antimicrobial

peptides, or novel high-specificity water filters coated with proteins binding specific contaminants. In another area, the toolkit

allows to create patterned cellulose structures in three dimensions (see Fig. 5a,b). In combination with functionalization, this

may be used in tissue engineering, for one-step production of cellulose based tissue engineering scaffolds that contain

specifically patterned growth factors. Furthermore, the remarkable robustness of K. rhaeticus within cellulose (see Fig. 1e)

offers potential uses in biosensing. The growing pellicle can store changes in environmental signals by writing the conditions

into different cellulose layers as they grow (see Fig. 5b), acting as a record akin to ice cores. Finally, as cellulose synthesis and

nitrogen fixation in closely related Acetobacteraceae are areas of active research, this toolkit is also a valuable resource for

basic studies aiming to dissect the molecular mechanisms of these processes. As there are many possible applications enabled

by this work, we are making this strain and the genetic engineering toolkit available through ATCC and AddGene.

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METHODS

Isolation, characterization and culturing of K. rhaeticus iGEM

K. rhaeticus iGEM was isolated from a Kombucha SCOBY (symbiotic colony of bacteria and yeast) of Czech origin (Happy

Kombucha, Eastbourne, UK) by streaking homogenized SCOBY material on HS-agarose (Supplementary Table S5), verifying cell

morphology under light microscope, and re-streaking isolated colonies on HS-agarose twice. Two percent (w/v) glucose was

used in HS unless stated otherwise. Glycerol stocks were prepared by culturing the iGEM strain statically in HS medium for 6

days, followed by addition of 0.2% (v/v) cellulase (T. reesei cellulase, cat. no. C2730; Sigma, St. Louis, USA), incubation at 230

rpm shaking, 30 °C for 1 day, addition of glycerol to 25% (v/v) and storage at -80 °C.

For cellulose production, seed cultures of K. rhaeticus iGEM were inoculated from glycerol stocks and grown statically at 30 °C

in Kombucha tea, HS or LGI medium (see Supplementary Table S5 and Supplementary Protocols for details). When grown with

shaking without cellulose production, cultures were grown in HS-cellulase (0.4% v/v) at 30 °C, 230 rpm shaking at 45° tube

angle. Unless otherwise stated, 50 mL Corning tubes (cat. no. CLS430829 SIGMA; Sigma) with 5-20 mL of media were used for

culturing. When grown on HS agar, plates were incubated inverted at 30 °C. For culturing of K. rhaeticus transformed with

plasmids encoding kanamycin or chloramphenicol resistance genes, kanamycin was added to 500 μg/mL for HS-agar and 50-100

μg/mL for liquid HS, and chloramphenicol added to 340 μg/mL for HS-agar and 34-68 μg/mL for liquid HS.

Cellulose productivity on different media was measured by culturing in 20 mL of HS-glucose (2% w/v), HS-sucrose (2% w/v), HS

without a carbon source (negative control) and Kombucha tea in 50 mL Corning tubes at 30 °C for 10 days, with loose caps for

increased air diffusion, and kept at 4 °C until measurement of cellulose weight (see Supplementary Protocols). Productivity of K.

rhaeticus iGEM was reported in comparison to productivity of the high-producing G. hansenii ATCC 53582 instead of maximal

cellulose yield per volume of media, as maximal total productivity is highly dependent on specific culturing conditions, and may

not be a good measure of genetically determined production capabilities. To test cellulose productivity on nitrogen-free LGI

medium, 80 μL of OD600=1 E. coli or K. rhaeticus iGEM seed culture was inoculated into 25 mL LGI medium in 50 mL glass tubes

(cat. no. 3119-0050; Thermo Fischer, Waltham, USA), and imaged 9 days post-inoculation.

For scanning electron micrographs, cellulose was gold-coated under vacuum and imaged at 2000-6000x magnification, 20kV.

See Supplementary Methods for details.

Chemical tolerance assays

For both E. coli and K. rhaeticus, cells were treated with 0.5 mL of 0.1 M NaOH, 0.1 M HCl, 70% ethanol, 10% bleach or PBS for

5, 30 or 90 minutes. Treatments were then plated; colonies photographed in white light and counted to determine the fraction

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of surviving cells compared to PBS-treated cells. For details of the assay, see Supplementary Methods.

Genome sequencing, assembly, bioinformatics and statistics

K. rhaeticus iGEM genome was sequenced with an Illumina MiSeq (Illumina) using 250 bp paired-end reads, to a coverage of

approximately 400x. Reads were then downsampled to 100x coverage, assembled using the BugBuilder pipeline (30), quality

controlled and annotated using Prokka (51). All statistical tests were performed with Prism 6 (GraphPad Software, Inc., La Jolla,

USA). For details of genome sequencing and bioinformatics, see Supplementary Methods.

Engineering of constitutive promoters, inducible promoters, CBD-fusions and sRNA construct

pSEVA331Bb and pSEVA321Bb were constructed from pSEVA331 and pSEVA321 respectively via substituting the native

polylinker with BioBrick prefix and suffix. This was done by PCR mutagenesis with Q5 polymerase (cat. no. M0491S; NEB, Hertz,

UK), primers i75, i76 (Supplementary Table S6), digestion with SpeI (cat. no. R3133S; NEB) and subsequent re-ligation.

Constitutive promoter-mRFP1 constructs (BBa_J23100 - Bba_J23117 by iGEM 2006 Berkeley) were received from the iGEM

Registry of Standard Biological Parts (52) and subcloned into pSEVA331Bb. ATc inducible constructs were kindly provided by Dr

Francesca Ceroni at Imperial College, and AHL inducible constructs BBa_J09855 (Jon Badalamenti, iGEM 2005) and BBa_F2620

(iGEM 2004 MIT) were received from Registry of Standard Biological Parts. Inducible constructs were then cloned into J23100-

mRFP1-pSEVA331Bb, replacing the constitutive J23100 promoter (thus controlling downstream mRFP1 expression) to create

pTet01, pTet02, pLux01 and pLux02.

CBDclos and CBDcex were received as BBa_K863111 and BBa_K863101 (iGEM 2012 Bielefeld) from the Registry of Standard

Biological Parts and CBDcipA and dCBD were synthesized as a GeneStrings (Life Technologies, Carlsbad, USA). CBDs were then

fused to sfGFP (BBa_I746909, iGEM 2007 Cambridge) and cloned into an expression vector (BBa_J04500, Kristen DeCelle iGEM

2005) downstream of pLacI promoter. sRNA construct was also synthesized as a GeneString (Life Technologies) based on

descriptions of Na. et al. (53) and subcloned downstream of the pLux promoter in pLux01, replacing mRFP1. All constructs were

first transformed into E. coli Turbo and colonies screened using colony PCR with GoTaq Green (cat. no. M7122; Promega,

Madison USA) and primers i53, i54 (see Supplementary Table S6). Plasmid DNA from positive colonies was extracted with

Qiagen QIAprep Spin Miniprep Kit (cat. no. 27104; Qiagen, Venlo, Netherlands), DNA sequenced (Source Biosciences,

Nottingham, UK), and correct sequences transformed into K. rhaeticus iGEM (see Supplementary Protocols). DNA sequences of

all constructs created in this work are accessible in the Registry of Standard Biological Parts (25) (see Supplementary Table S4

for accession numbers). As many of the constructs characterized in K. rhaeticus iGEM are widely used and accessible through

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the Registry of Standard Biological Parts, no constructs were codon optimized, in order to allow them to be used in K. rhaeticus

iGEM without modification by the user.

Characterization of constitutive promoters, inducible promoters, CBD-fusions and sRNA construct

For characterization of constitutive promoters and inducible promoters without cellulose formation, 1 - 5 mL of liquid HS-

cellulase (8-10% v/v) containing 34 μg/mL chloramphenicol in 50 mL Corning tubes were inoculated from seed cultures to

OD600=0.02, grown at 30 °C, 230 rpm for 3 h and 200 μL of culture was then pipetted into 96-well plates (Corning Costar, New

York USA). Inducer was added to inducible cultures to 1 μg/mL for ATc and 1 μM for AHL. High concentrations of cellulase were

used to remove any interference by cellulose on spectroscopy measurements. Plates were covered with Breathe-Easy

membrane (cat. no. Z380059; Sigma), and OD600 and mRFP1 intensity (excitation 590 nm, emission 630 nm) were measured

every 15 minutes using Synergy HT microplate reader (BioTek, Winooski USA) at 29 °C, high shaking speed.

For characterization of inducible promoters in pellicle form (Fig. 3d,f and Supplementary Fig. S8), K. rhaeticus iGEM containing

pLux01 was inoculation from glycerol stocks into HS with 34 μg/mL chloramphenicol and grown statically for 8 days at 30 °C. For

cultures induced during growth, AHL was added to 1 μM before inoculation, and for cultures induced in pellicle, AHL was added

to 1 μM 6-days post inoculation. Pellicles were washed with PBS 8 days after inoculation, microscopy samples prepared with a

sterile razor, and fluorescence quantified using fluorescence microscopy (Nikon Eclipse Ti; Nikon, Tokyo, Japan) at mCherry pre-

set (590nm emission, 0.2 s exposure, 675V EM gain, 150x magnification). A single layer cellulose sheet was located on the

sample and fluorescence intensity determined as intensity/exposure time for quantitative measurements. For white-light

images (Fig. 3e), 250 mL beakers (cat. no. CLS1000250; Sigma) containing 100 mL of HS media were inoculated with 5 mL of

OD600=1 seed culture, the beakers covered with Breathe-Easy membrane, and beakers incubated statically at 30 °C. For the

induced pellicle, 1000 μL of 100 mM AHL was pipetted daily 4 days after inoculation along the edges for the pellicle, and pellicle

imaged 9 days post-inoculation.

For characterization of CBD binding strengths, 200 μL of CBD-sfGFP fusion proteins extracted from E. coli (see Supplementary

Protocols) were added to a 96-well plate containing homogenized bacterial cellulose, incubated overnight at 4 °C and washed

thrice with treatment (dH2O, PBS, 5% BSA or 70% EtOH; see Supplementary Protocols). GFP fluorescence was measured on a 96-

well plate reader (Synergy HT, Biotek; see Supplementary Protocols for details).

For characterization of sRNA construct, HS with 34 μg/mL chloramphenicol was inoculated to OD600=0.04 with K. rhaeticus

iGEM containing plasmid J09855-sRNA-331Bb, and induced with 10-500 nM AHL. 40 h after inoculation, pellicles were washed,

dried at 60 °C for 16 h and weighed (see Supplementary Protocols for details). For characterization of growth rate, OD600 was

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measured in (3% v/v) HS-cellulase with 34 μg/mL chloramphenicol using the protocol use for characterization of constitutive

promoters.

Engineering of functionalized biomaterials

For spatial patterning, K. rhaeticus containing pLux01 was inoculated into 1 L HS chloramphenicol in 2 L Erlenmeyer flasks, 500

μL of 100 nM AHL was added to one side of the pellicle 2 days later, and pellicle was imaged 4 days after induction. For

temporal patterning, 250 mL beakers with 100 mL of HS media were inoculated with 5 mL of seed culture at OD600=1, the

beakers covered with Breathe-Easy membrane, and incubated statically at 30 °C. After this, 1000 μL of 100 mM AHL was

pipetted along the edges of the pellicle daily with membrane replacement, starting at different days post-inoculation as shown

in Fig. 5b, and pellicle imaged 9 days post-inoculation. For functionalization of cellulose with mRFP1 and CBDcipA-sfGFP,

proteins were first produced in E. coli, extracted via sonication, and extracts applied to purified wet or dried bacterial cellulose

respectively. For mRFP1-functionalized cellulose, fluorescence intensity was determined from a single cellulose sheet using

fluorescence microscopy. For CBDcipA-sfGFP, extracts were applied to dried bacterial cellulose with a paintbrush and dried. The

resulting material was then crafted into accessories by a professional fashion designer. See Supplementary Protocols for further

details.

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ACKNOWLEDGEMENTS

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We thank Dr Cheng-Kang Lee from National Taiwan University of Science and Technology and Dr Jyh-Ming Wu from Chinese

Culture University for sharing plasmid pBla-Vhb-122, and Ms Victoria Geaney from the Royal College of Art, London for help

with functionalized garments. At Imperial College London, we thank Dr Koon-Yang Lee for helpful advice and feedback on the

manuscript, Dr Takayuki Homma for help with genome library preparation, Dr Laurence Game for help and advice with genome

sequencing, Mr Geraint Barton for help and advice with genome assembly and analysis, Dr Francesca Ceroni for providing ATc-

inducible constructs and advice, and Ms Catherine Ainsworth, Mr Nicolas Kral, Dr Geoff Baldwin and Dr Guy-Bart Stan for

instruction and helpful discussions throughout the project. Finally, we thank Mahmoud Ardakani (Imperial College London) for

taking SEM images and Dr Carlos Bricio Garberi (Imperial College London) for taking pellicle photographs.

AUTHOR CONTRIBUTIONS

M.F., H.H., and B.R., isolated and characterized the iGEM strain; M.F. and B.R. sequenced the genome; M.F. and J.A. scaffolded

and analysed the genome; M.F., H.H., G.S., C.N.M., X.S.M., L.A.G. and D.P. created the genetic engineering toolkit; M.F. created

the sRNA construct; C.N.M., G.S., X.S.M. and H.H. created CBD fusion proteins; M.F., G.S., H.H., X.S.M. and C.N.M. created

patterned and functionalized biomaterials; H.H, G.S, T.E. and M.F. conducted temporal patterning experiments, B.R., G.S.,

C.N.M., H.H., X.S.M. and L.A.G. created sfGFP-functionalized garments; M.F., C.L., D.K., C.N.M., D.P. and H.C. analysed data;

M.F. and T.E. wrote the manuscript; T.E., B.R., P.F., R.K. and K.J. supervised the work; all authors contributed to planning the

study.

COMPETING FINANCIAL INTERESTS

H.H. and G.S. are researching the industrial uses of cellulose functionalized with CBD fusion proteins as part of CustoMem Ltd.

FIGURE LEGENDS

Figure 1. Characterization of Komagataeibacter rhaeticus iGEM (a) Morphology of a typical cellulose pellicle produced by K.

rhaeticus iGEM. White patches are light reflected by the pellicle surface. (b) Cellulose productivity of K. rhaeticus iGEM and G.

hansenii ATCC 53582 on different growth media, shown as pellicle dry weight after 10-day incubation in 20 mL liquid HS media.

Cellulose production of K. rhaeticus exceeds that of G. hansenii in sucrose-containing media (HS-sucrose and Kombucha tea,

adjusted p=0.0128 and p=0.039 respectively), but is lower than that of G. hansenii in HS-glucose (adjusted p=0.027). (c-d)

Growth and production of a cellulose pellicle (denoted by arrow) by K. rhaeticus iGEM in nitrogen-free LGI medium. K. rhaeticus

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iGEM grows significantly compared to negative controls G. hansenii and E. coli (adjusted p=0.026 and p=0.011 respectively),

while G. hansenii and E. coli do not differ (adjusted p=0.742) (e) Comparison of K. rhaeticus and E. coli survival after incubation

for 5, 30 or 90 minutes with toxic chemicals (0.1 M HCl, 0.1 M NaOH, 70% EtOH, 10% bleach). Survival is defined as fraction of

survived cells compared to PBS-treated cells. (f) Scanning electron micrographs of K. rhaeticus iGEM encased in bacterial

cellulose, taken after 8 days of growth at 6000x magnification. N=3 biological replicates for all experiments, error bars=SD.

Statistical significance determined for (b) with 2-way ANOVA and Bonferroni’s multiple comparisons test and for (d) 1-way

ANOVA with Tukey’s multiple comparisons tests. For (a) and (c), images were taken 9 days post-inoculation. Images were

cropped and contrast was adjusted to improve clarity, without affecting details. See Methods for experimental details.

Figure 2. K. rhaeticus iGEM genome. (a) Overview of K. rhaeticus iGEM genome. iGEM genome totals 3.87 Mbp with a GC% of

62.7, and contains a predicted 3505 protein coding genes, 3 rRNAs, 52 tRNAs and 13 other non-coding RNAs. The genome

consists of a chromosome of approximately 3.16 Mbp and at least two plasmids – pKRi01 (238 kbp) and pKRi02 (3 kbp). The

chromosome contains 2899 predicted genes with 63% GC content and contains 4 copies of acs (cellulose synthase) operons. For

the chromosome, consecutive rings show (from outside in): (1) read coverage, (2) genes on forward and (3) reverse strands, (4)

acs operons involved in cellulose synthesis, (5) GC% and (6) GC skew. Additionally the genome contains 37 scaffolds (totalling

460 kbp) that could not be confidently placed due to repetitive sequences. These scaffolds may be part of the chromosome,

plasmids, or may belong to additional plasmids (closely related species G. xylinus NBRC 3288 and K. xylinus E25 contain 5 and 7

plasmids respectively). (b) Phylogenetic relationships and (c) amino acid sequence identity of acs cellulose synthase operons.

Phylogeny and sequence identity indicate that acs2 and acs4 operons are most closely related. Amino acid sequences were

aligned and percent identity calculated using MUSCLE (54) and the tree was generated using the Neighbour-Joining method

(55). The tree is drawn to scale, with Bootstrap values(56) from 1000 replicates shown next to the branches. All positions

containing gaps were eliminated from analysis. See Methods for further details on sequencing and analysis.

Figure 3. K. rhaeticus synthetic biology tookit (a) Overview of the toolkit contents. (b) Constitutive promoter strengths and (c)

AHL and ATc inducible construct expression strengths in K. rhaeticus iGEM, as measured by total mRFP1 fluorescence per cell

(fluorescence at 630nm divided by OD600). Promoter strengths in (b) and (c) were assayed in liquid HS-cellulase, to remove

formation of cellulose fibrils interfering with measurements. (d) Total mRFP1 fluorescence expressed from pLux01 construct,

when K. rhaeticus cells were induced with AHL inside cellulose pellicle (Induced in pellicle), compared to uninduced or wild-type

(WT) cells. Induction caused a significant increase in fluorescence compared to non-induced or wild-type cells (p<0.001 for both

induced vs non-induced, and induced vs wild-type, determined with 1-way ANOVA and Tukey’s post-hoc tests). Cells were

grown in HS (without cellulase) and fluorescence quantified by fluorescence microscopy image analysis. (e) Induction in pellicle

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results in visible mRFP1 production compared to uninduced cells (indicated with the white arrow, also see Supplementary Fig.

S8), and (f) results in granular fluorescence due to localization within cells. N=3 for all experiments, error bars=SD. For (e and f),

images were cropped and contrast was adjusted to improve clarity. See Supplementary Fig. S7 for detailed overview of

constructs and Methods for details of characterization assays.

Figure 4. An sRNA construct (J-sRNA-331Bb) for control of cellulose production. (a) Overview of the sRNA silencing construct.

Constitutively produced LuxR binds to pLux in the presence of AHL and upregulates production of E.coli Hfq and an sRNA

targeting the UGPase mRNA. The 5’ end of the sRNA contains a 24 bp sequence complementary to UGPase mRNA, and binds to

it in the presence of E. coli Hfq (53), leading to silencing of the UGPase gene. (b) Cellulose production of induced and uninduced

cultures shown as cellulose dry weight measured 40 h post-inoculation. “No cellulose” = empty weighing boats, “pSEVA331Bb

100 nM AHL” and “pSEVA331Bb” = iGEM strain with empty pSEVA331Bb vector with and without 100 nM AHL, respectively. Full

induction with 100 nM and 500 nm AHL results in complete suppression of cellulose synthesis (adjusted p<0.0001 both

“Uninduced” vs “100 nM AHL” and “500 nM AHL”). Addition of AHL itself does not decrease cellulose productivity (adjusted

p>0.999 for “pSEVA331Bb” vs “pSEVA331Bb with 100nM AHL”) and uninduced cells are not different from negative controls

(adjusted p=0.12 for “Uninduced” vs “pSEVA331Bb 100nM AHL”). (c) OD600 of cultures 3 h post-inoculation. Differences

between OD600 are not significant for any comparisons (adjusted p>0.05), showing that induction of the sRNA silencing

construct does not reduce growth rate. N=5 for all samples (except N=3 for (b) pSEVA331-Bb and pSEVA331-Bb with 100 nM

AHL), Statistical significance determined with 1-way ANOVA and Tukey’s multiple comparison tests for (b) and (c). See Methods

for further details of all assays.

Figure 5. Engineering of patterned and functionalized cellulose materials on macroscale. (a) Spatial patterning. Cells were

induced for mRFP1 expression by addition of 100 nM AHL to one side of a 1 L culture and the pellicle imaged for red

fluorescence 3 days post-induction. Inset shows the pellicle imaged in white light. (b) Temporal patterning. Cells were induced

with AHL daily at different times through pellicle growth (0 days only, or starting at 0, 2, or 4 days post-inoculation), and imaged

9 days post-inoculation, with overview (i), white-light (ii) and fluorescence (iii) images of the pellicles and pellicle cross-sections

shown. Note that pellicles induced on day 0 only show low fluorescence due to natural degradation of AHL in the media over

time. (c) Overview of cellulose functionalization strategy via post-hoc addition of mRFP1 extracted from E. coli. (d) Fluorescence

microscopy of a cross-section of cellulose functionalized with mRFP1 through addition of mRFP1 extracted from E. coli. Smooth

fluorescence is seen throughout the pellicle cross-section, compared to granular fluorescence seen in Fig. 3e (also see

Supplementary Fig. S11). For (a) and (b iii), computationally determined and averaged brightness (Grey Value) along the pellicle

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cross-section is shown correspondingly to the right of the image. Images were cropped and contrast was adjusted to improve

clarity for all images. See Methods for fluorescence imaging conditions and other details.

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