<p>We initially researched the enzymatic pathway for Bacterial cellulose in *Komagataeibacter*. 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 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. <!--to be Add original plasmid maps for in- silico cloned knockouts--></p>
<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 for the constructs with PaqcI overhangs to later assemble.In addition we amplified the Level 0 backbone pSB1C30 with a Chloramphenicol resistance cassette and PaqcI to act as our backbone. And an ampicillin resistance cassette to select for later in final transformation in *K. xylinus*.</p>
<p>After the 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 transformed in *E. coli* based on our DH5α transformation Protocol and plated on LB agar plates with both Cam and Amp inside for selection </p>
<p>Add planned Plasmid maps</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 3 of them were successful in growing colonies and initial colony PCR result showed that full plasmid was inside them</p>
<p>To verify the plasmid sequence. We did full plasmid sequencing through next generation Nanopore sequencing by Microsynth. </p>
<p>Add Sequencing results for knockouts</p>
<p>Sequence results showed that one of the knockouts actually was very similar to the planned plasmid we designed but in CD014 a homology region was missing. We are currently unsure why exactly. It was slightly unfortunate as both inducible constructs that were sequenced share the region affected but it may be due to human error during assembly. Due to time constraints a repetition of the transformation was not an option and we continued with CD015 plasmid construct</p>
<p>After verifying results through sequencing. We then transformed the previously prepared electrocompetent *K. xylinus* with the plasmid CD015. After 5 days visible colonies formed on plates</p>
<p>Add pictures of *K. xylinus* plate CD015 </p>
<p>Colonies for cPCR results were picked and resulting gel indicate that knockout through homologous recombination was probably successfully integrated into the genome of *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>
<!--Add planned Plasmid maps-->
<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>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>
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>
<p>Although the test is qualitative in nature it was mainly to assess if Bacterial cellulose is visible if induced and if so if there is a visual difference between performance of WT and knockout. </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> After Wiki freeze </p>
<h2>Learn</h2>
<p>Uwe were able to achieve a viable transformation and through </p>
<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>
