<p>We initially researched the enzymatic pathway for Bacterial cellulose in *Komagataeibacter xylinus*. In literature we found that the responsible genes BcsA, BcsB, BcsC and BcsD are the main enzymes involved in the synthesis of Bacterial cellulose. However, other Enzymes including BcsZ and BcsH also are necessary to ensure stable production. In addition we designed our primers to have PaqcI restriction sites so that we were able to use an golden gate assembly approach. <!--to be Add original plasmid maps for in- silico cloned knockouts--></p>
<p>We initially researched the enzymatic pathway for Bacterial cellulose in *Komagataeibacter xylinus*. In literature we found that the responsible genes BcsA, BcsB, BcsC and BcsD are the main enzymes involved in the synthesis of Bacterial cellulose. However, other Enzymes including BcsZ and BcsH also are necessary to ensure stable production. In addition we designed our primers to have PaqcI restriction sites so that we were able to use an golden gate assembly approach. </p>
<!--to be Add original plasmid maps for in- silico cloned knockouts-->
<h2>Build</h2>
<p>We assembled our constructs by firstly by starting a genome DNA extraction in *K. xylinus* to have a DNA template to amplify the necessary homology regions as well as an ampicilin ressistance cassete for the planned constructs with PaqcI overhangs to later assemble.In addition we also amplified the Level 0 backbone pSB1C30 that already has a chloramphenicol resistance cassette with primers that add PaqcI restriction site overhangs to act as our backbone. So that in the end two antibiotics could be used for final selection in *K. xylinus*.</p>
<p>We assembled our constructs by firstly by starting a genome DNA extraction in *K. xylinus* to have a DNA template to amplify the necessary homology regions as well as an ampicilin ressistance cassete for the planned constructs with PaqcI overhangs to later assemble. In addition we also amplified the Level 0 backbone pSB1C30 that already has a chloramphenicol resistance cassette with primers that add PaqcI restriction site overhangs to act as our backbone. So that in the end two antibiotics could be used for final selection in *K. xylinus*.</p>
<p>After the PCR amplification of every fragment was complete. A Golden gate assembly with PaqcI and T4 ligase was done to assemble our constructs. After completion, our constructs were then transformed in *E. coli* based on our DH5α transformation Protocol and plated on LB agar plates with both chloramphenicol and ampicilin inside for selection </p>
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<p>Unfortunately some of the plates appeared to have dried out in the incubator they were placed in. Still out of 4 planned knockouts golden gates 3 of them were successful in growing colonies and initial colony PCR result showed primer binding on homology as well as ampicillin ressistance cassete in roughly the predicted basepair length.</p>
<p>*E. Coli* DH5α transformed with CD015 pSB1C30 ΔbcsZH AmpR colonies on LB agar plates with both ampicilin and chloramphenicol added for selection.</p>
<p>Unfortunately some of the plates appeared to have dried out in the incubator they were placed in. Still out of 4 planned knockouts golden gates 2 of them were successful in growing colonies and initial colony PCR result showed primer binding on homology as well as ampicillin ressistance cassete resulted in PCR product amplification roughly in the predicted basepair length.</p>
<p>Picture : Gel picture of cPCR results showing visible bands at predicted length for assembled constructs CD031 and CD015 </p>
<p>Before further transformation in *K. xylinus* we wanted to verify the plasmid sequences. Therefore We did whole plasmid sequencing through next generation nanopore sequencing by Microsynth. </p>
<!--Add Sequencing results for knockouts-->
<p>Sequence results showed that only one of the knockout candidates CD015 pSB13C0 delta bcsH delta bcsZ was succesfully assembled. We are currently unsure why exactly. It was slightly unfortunate as both inducible constructs that were sequenced as well share the same affected homology region affected but due to time constraints a repetition of the transformation was not an option and we continued with CD015 plasmid construct</p>
<p>We then transformed *K. xylinus* with the plasmid CD015 through electroporation. After 5 days visible colonies formed on plates</p>
<!--Add pictures of *K. xylinus* plate CD015 -->
<p>Colonies for cPCR were picked and resulting PCR and gel electrophoresis indicate that knockout of BcsH and BcsZ through homologous recombination was successfully integrated into the genome of *K. xylinus*</p>
<p>Sequence results showed that only one of the knockout candidates CD015 pSB13C0 ΔbcsH ΔbcsZ was succesfully assembled. We are currently unsure why exactly. It was slightly unfortunate as both inducible constructs that were sequenced as well share the same affected homology region affected but due to time constraints a repetition of the transformation was not an option and we continued with CD015 plasmid construct</p>
<p>We then transformed *K. xylinus* with the plasmid CD015 through electroporation. After 5 days visible colonies formed on YPD agar plates.</p>
<p>Picture: *K.xylinus* CD015 colonies on YPD agar plate with 0.2% cellulase as well as ampicilin and chloramphenicol for selection. After 6 days of cultivation visible colonies were circled on plate to use for cPCR to veify success of transformation.</p>
<p>Colonies for cPCR were picked and resulting PCR and gel electrophoresis indicate that knockout of the gene *BcsH* and *BcsZ* through homologous recombination was successfully integrated into the genome of *K. xylinus*</p>
DNA electrophoresis gel pic. Layout of gel pic from left to right: ladder,2-7 replacing native constitutive promoter for the Bcs ABCD with paraBAD /inducible arabinose promoter and AraC and AraE genes through homologous recombination, 8-13 knockout of BcszH region through homologous recombination, 14-15 replacing native constitutive promoter for the Bcs ABCD with paraBAD /inducible arabinose promoter and AraC genes through homologous recombination.</p>
<h2>Test</h2>
<p>After positive control we really wanted to immediately characterise our transformed strains.
However, due to time constraints we were not able to achieve an in depth characterisation. However, we still managed to do an initial comparative inoculation test by Preparing SOC media with 2% glucose and 1% Arabinose as well as SOC media with just 2% glucose added. We then added colonies from knockout strain CD015, inducible strain CD027 and WT for comparison.</p>
<!--Add pictures of *K. xylinus* knockout and WT Well-plate -->
<p>Although the test is qualitative in nature it was mainly to assess if bacterial cellulose production in the knockout is visually worse than the wild type strain. After </p>
<p>Picture: *K.xylinus* CD015 colonies were used to inoculate a 6-wellplate based on layout (left). A: shows the plate after initial inoculation. B: shows the well-plate after 3 days. C: shows the well plate after 9 days of standing cultivation in room temperature.</p>
<p>Although the test is qualitative in nature it was mainly to assess if bacterial cellulose pellilce formation in the knockout is visually worse than the wild type strain. After 3 and 9 days no bacterial cellulose pellilce was seen indicating that loss of function was succesfully engineered in *K. xylinus* CD015 strain </p>
<h2>Learn</h2>
<p>We were able to achieve a viable transformation and through initial inoculation testing we were able to show that the strain indeed does not produce a bacterial cellulose pellicle even after 9 days of cultivation. furthermore through growth rate experiments we were able to show that the growthrate of the knockout strain is higher. This proves the pedictions made with our metabolic model where bacterial cellulose production slows down growth of K. xylinus. Further experiments for future iGEM teams continuing our work may be to assemble and transform the remaining constructs targeting different or all regions in the bacterial cellulose synthase complex in *K. xylinus* to see if they prove viable as well and if increased growthrate compared to Wildtype strain stays consistent. </p>
<p>1. Römling, U., & Galperin, M. Y. (2015). Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions. Trends in microbiology, 23(9), 545–557. https://doi.org/10.1016/j.tim.2015.05.005</p>
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<p>The cause could be that the rbs in combination with the araC and araE genes were stressing the E. coli as it increased the AraC amount in the cell. Resulting in the consistent removal of the ribosome binding site by the E. coli where the AraC gene was present as well. Luckiliy for us we had overhangs that were identical to the startcodon plus another base. Therefore there was a low chance it binds incorrectly overgoing the rbs. While we were lucky in that case future iGEM Teams should definitely not count on that and take this definitely into consideration if they want work with the construct</p>
<p>1. Römling, U., & Galperin, M. Y. (2015). Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions. Trends in microbiology, 23(9), 545–557. https://doi.org/10.1016/j.tim.2015.05.005
2. Mangayil, R., Rajala, S., Pammo, A., Sarlin, E., Luo, J., Santala, V., Karp, M., & Tuukkanen, S. (2017). Engineering and Characterization of Bacterial Nanocellulose Films as Low Cost and Flexible Sensor Material. ACS applied materials & interfaces, 9(22), 19048–19056. https://doi.org/10.1021/acsami.7b04927</p>
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<p> As final update experiment, we were interested in further investigating differences in cell viability between all engineered strains. To achieve this, we measured the OD Growth of both the knockout and inducible BC K. xylinus strain in a microplate reader. </p>
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<summary><b> Outlook </b></summary>
<h2>Outlook</h2>
<p>With that we could eventually reduce the need for cellulase necessary to increase cell count and optimise carbon intake by targeting knockouts or overexpression for different genes and or clone an inducible promoter in front of the gene we want to up or down regulate, giving us even more control over the Bacterial cellulose synthesis. Through this we hope to push the scale in our favour to achieve a true sustainable product.</p>
<p>source</p>
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Sources:
1. Römling, U., & Galperin, M. Y. (2015). Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions. Trends in microbiology, 23(9), 545–557. https://doi.org/10.1016/j.tim.2015.05.005
2. Mangayil, R., Rajala, S., Pammo, A., Sarlin, E., Luo, J., Santala, V., Karp, M., & Tuukkanen, S. (2017). Engineering and Characterization of Bacterial Nanocellulose Films as Low Cost and Flexible Sensor Material. ACS applied materials & interfaces, 9(22), 19048–19056. https://doi.org/10.1021/acsami.7b04927
## Dye Group
To achieve our goal to have coloured bacterial cellulose mats, we went through multiple design cycles in order to come closer to achieving a modular colouring system.
