@@ -114,72 +114,115 @@ Note: All pictures and tables above are made by team members and are saved in ou
## Inducible BC
To achieve our goal to have controlled activation of the Bacterial cellulose synthesis apparatus in Komagataeibacter xylinus. We went through multiple design cycles to achieve a genetically engineered strain where the bacterial cellulose production can only be activated by the presence of arabinose sugar.
To achieve our goal to have controlled activation of the bacterial cellulose synthesis apparatus in *Komagataeibacter xylinus*. We went through multiple design cycles to achieve a genetically engineered strain where the bacterial cellulose production can only be activated by the presence of arabinose sugar. Furthermore, by knocking out specific parts of the bacterial cellulose gene complex we can potentially investigate the metabolic cost by measuring the growth rate of *K. xylinus* and comparing it to the wildtype.
<!--Start Collaps section Cycle 1 inducible BC knockout-->
<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 *K. xylinus*. In literature, we found that the responsible genes *bcsA*, *bcsB*, *bcsC* and *bcsD* are expressing for the main enzymes involved in the synthesis of bacterial cellulose. Sequentially other genes like *bcIX*, *bcsZ* and *bcsH* express for enzymes that are also essential to ensure stable bacterial cellulose production as those are part of the regulatory process and the secretory machine in bacterial in *K. xylinus*(McNarma JT, et al.,2015; Römling U., et al.,2015).</p>
<p><b>Figure 1</b>: Enzymatic pathway map highlighting the enzymes responsible in the bacterial cellulose synthesis, secretion and regulation apparatus. Graphic originally from McNarma JT, et al., 2015. A molecular description of cellulose biosynthesis. Annual review of biochemistry, 84, 895–921. https://doi.org/10.1146/annurev-biochem-060614-033930.</p>
<p>As it is currently not known by how many operons regulate all genes, but all genes are located next to each other, we decided to focus on the knockout of specific genes and specific gene clusters that based on the literature, are regulated by the same operon in other bacterial cellulose producing strains: 1. *bcIX*; 2. *bcsA*, *bcsB*, *bcsC*, *bcsD*; 3. *bcsZ*, *bcsH*; and 4. the complete knockout of all afformentioned genes.</p>
<p>To achieve the planned knockouts in *K. xylinus*, we designed our constructs (<b>Figure 3-6</b>) with homology regions surrounding the targeted gene clusters. Furthermore we specifically went with the pSB1C30 backbone, because the ori sequence is compatible with *K. xylinus.* Thirdly, we also added an ampicillin ressistance cassete in addition to the chloramphenicol resistance casette already present in the backbone to increase the likely hood that selected colonies include the planned constructs. Finally, to assemble our constructs we decided to use a Golden Gate assembly aproach with PaqcI and therefore designed the primers used for amplification of all necessary fragments to have PaqcI restriction site overhangs.</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 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>
<p>We assembled our constructs by firstly starting a genome DNA extraction in *K. xylinus* to have a DNA template to amplify all the necessary homology regions. Sequentially, we also amplified a ampicilin ressistance cassete and the pSB1C30 vector backbone that already has a chloramphenicol resistance cassette present. So that in the two antibiotics could be used for selection in *E. coli* DH5α*K. xylinus*.</p>
<p><b>Figure 7</b>: Screenshot of Overview table used to document PCR amplification for all constructs planned for transformation in *K. xylinus.*</p>
<p>After PCR amplification of every fragment was complete, a Golden gate assembly with PaqcI and T4 ligase was done to assemble all our constructs. After completion, our presumed assembled constructs were then transformed in *E. coli* DH5α based on our DH5α transformation protocol and then plated on LB agar plates with both chloramphenicol and ampicillin for selection. </p>
<p><b>Figure 8</b>: *E. coli* DH5α transformed with CD015 pSB1C30 Δ*bcsZH amp*. Overnight, visible colonies formed on LB agar plates with both ampicillin and chloramphenicol.</p>
<p>Unfortunately, some of the plates appeared to have dried out in the incubator after one day, they were placed in. Still, out of four planned knockout constructs, two of them were successful in growing colonies. Colony PCR with primers binding on the vector backbone and the ampicillin resistance cassete, showed visible bands in colonies transformed with CD015 pSB1C30 Δ*bcsZH amp* and colonies transformed with CD031 pSB1C30 Δ*bcsABCDHZ*Δ*bcIX amp*.</p>
<p><b>Figure 9</b>: Gel picture of colony PCR results showing visible bands at predicted length for colonies transformed with assembled construct CD015 pSB1C30 Δ*bcsZH amp*(middle) and colonies transformed with assembled construct CD031 pSB1C30 Δ*bcsABCDHZ*Δ*bcIX amp* (right). </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>
<p>Sequence results showed that only one of the knockout candidates CD015 pSB13C0 Δ*bcsH*Δ*bcsZ*, was successfully 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 pSB1C30 Δ*bcsZH amp*.</p>
<p>We transformed *K. xylinus* with CD015 pSB1C30 Δ*bcsZH amp* through electroporation. After 5 days, visible colonies formed on YPD agar plates with ampicillin and chloramphenicol.</p>
<p><b>Figure 10</b>: *K. xylinus* CD015 pSB1C30 Δ*bcsZH amp* colonies on YPD agar plate with 0.2% cellulase as well as ampicillin and chloramphenicol for selection. After 5 days of cultivation visible colonies were marked on plate to use for cPCR to verify 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>
<p><b>Figure 11</b>: DNA electrophoresis gel pic. Layout of gel pic from left to right: ladder,2-7 replacing native constitutive promoter for the *bcsABCD* 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 *bcsABCD* 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> After Wiki freeze </p>
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. We then added colonies from knockout strain CD015, inducible strain CD027 and WT for comparison.</p>
<p><b>Figure 12</b>: *K. xylinus* CD015 colonies were used to inoculate a 6-wellplate based on layout (left). <b>A</b>: shows the plate after initial inoculation. <b>B</b>: shows the well-plate after 3 days of standing cultivation in room temperature. <b>C</b>: 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 pellicle formation in the knockout is visually worse than in the wild type strain. After 3 and 9 days no bacterial cellulose pellilce was seen in the knockout strain, indicating that loss of function was succesfully engineered in *K. xylinus* CD015 pSB1C30 Δ*bcsZH amp* 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>
<p>We were able to achieve a viable transformation and through initial inoculation testing we were able to show that the strain does indeed not produce a bacterial cellulose pellicle even after 9 days of cultivation. Furthermore, through growth rate experiments we were able to show that the growth rate of the knockout strain is higher. This matches the predictions made with our metabolic model, where bacterial cellulose production slows down growth in *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 for a complete knockout of the bacterial cellulose synthesis function or if potentially a knock down effect can be perceived. Finally, it would also be of interest to compare the growth rate in all of the growth rate in all of the knockout strains to better understand the metabolic cost of each gene in *K. xylinus*</p>
</details>
<!--End Collaps section-->
<!--Start Collaps section Cycle 2 inducible BC strain-->
<p>We initially researched the enzymatic pathway for Bacterial cellulose in Komagataeibacter. In literature we found that the Enzymes 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.</p>
<p>We initially researched the enzymatic pathway for bacterial cellulose in *K. xylinus*. In literature, we found that the responsible genes *bcsA*, *bcsB*, *bcsC* and *bcsD* are expressing for the main enzymes involved in the synthesis of bacterial cellulose. Sequentially other genes like *bcIX*, *bcsZ* and *bcsH* express for enzymes that are also essential to ensure stable bacterial cellulose production as those are part of the regulatory process and the secretory machine in bacterial in *K. xylinus*(McNarma JT, et al.,2015; Römling U., et al.,2015).</p>
<p><b>Figure 1</b>: Enzymatic pathway map highlighting the enzymes responsible in the bacterial cellulose synthesis, secretion and regulation apparatus. Graphic originally from McNarma JT, et al., 2015. A molecular description of cellulose biosynthesis. Annual review of biochemistry, 84, 895–921. https://doi.org/10.1146/annurev-biochem-060614-033930.</p>
<p>As it is currently not known by how many operons regulate all genes, but all genes are located next to each other, we decided to focus on replacing the constitutive promoter for specific genes and specific gene clusters that based on the literature, are regulated by the same operon in other bacterial cellulose producing strains: 1. *bcIX*; 2. *bcsA*, *bcsB*, *bcsC*, *bcsD*; 3. *bcsZ*, *bcsH*.</p>
<p>To achieve the planned exchange of the native constitutive promoters in *K. xylinus* with an inducible promoter, we designed our constructs (<b>Figure 3-8</b>) with homology regions surrounding the targeted gene clusters. Furthermore we specifically went with the pSB1C30 backbone, because the ori sequence is compatible with *K. xylinus.* Thirdly, we also added an ampicillin ressistance cassete in addition to the chloramphenicol resistance casette already present in the backbone to increase the likely hood that selected colonies include the planned constructs. In a Finally, to assemble our constructs we decided to use a Golden Gate assembly aproach with PaqcI and therefore designed the primers used for amplification of all necessary fragments to have PaqcI restriction site overhangs.</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><b>Figure 9</b>: Table overview of necessary PCRs for both knockout and inducible BC</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 DH5 alpha transformation Protocol and plated on LB agar plates with both Cam and Amp inside for selection </p>
<p><b>Figure 10</b>: *E. Coli* DH5α transformed with CD027 pSB1C30 *arac am araE paraBAD bcsA-D amp* colonies on LB agar plates with both ampicilin and chloramphenicol.</p>
<p>Unfortunately some of the plates appeared to have dried out in the incubator they were placed in. Still out of 6 planned inducible constructs golden gates 3 of them were successful in growing colonies and initial colony PCR result showed that part of plasmid full plasmid was inside them</p>
<p><b>Figure 11</b>: Gel picture of cPCR results showing visible bands at predicted length for assembled constructs CD017, CD018 and CD027. Fragment corresponding to CD028 was smaller than predicted and therefore not taken into further consideration </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 two of the inducible BC constructs showed almost identical sequences very similar to the planned plasmid we designed CD017 and CD027. Due to time constraints a repetition of the transformation was not an option and we continued with the named plasmid constructs</p>
<p>After verifying results through sequencing. We then transformed the previously prepared electrocompetent *K. xylinus* with the plasmid CD017 and CD027. 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>
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>
<p>Sequence results showed that two of the inducible BC constructs showed almost identical sequences for the planned plasmids CD017 and CD027. However, due to time constraints, a repetition of the transformation was not an feasible option and we continued with the verified plasmid constructs</p>
<p>After verification through sequencing, we then transformed the previously prepared electrocompetent *K. xylinus* dsm 2325 strains with the plasmid CD017 and CD027. After 5 days visible colonies formed on plates</p>
<p>Colonies for cPCR results were then picked and resulting gel indicate that knockout through homologous recombination was probably successfully integrated into the genome of K. xylinus</p>
<p><b>Figure 11</b>: DNA electrophoresis gel pic. Layout of gel pic from left to right: ladder,2-7 replacing native constitutive promoter for the *bcsABCD* 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 *bcsABCD* 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><b>Figure 13</b>: *K.xylinus* CD027 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>While we were ultimately succesful in generating a viable inducable *Komagataeibacter* strain. There were several points during the in silico cloning as well as the wetlabstage where improvement is indeed possible. The whole plasmid sequencing showed that in all *E. coli* with the inducible constructs the rbs was missing according to sequencing results</p>
<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>
<p><b>Figure 15</b>: picture showing sequence alignment of CD017 </p>
<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>
</details>
<!--End Collaps section-->
<!--Start Collaps section inducible BC outlook -->
<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>
</details>
<!--End Collaps section-->
<!--Start Collaps section inducible BC outlook -->
...
...
@@ -187,10 +230,44 @@ To achieve our goal to have controlled activation of the Bacterial cellulose syn
<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>
</details>
<!--End Collaps section-->
Sources:
1. McNamara, Joshua T et al. “A molecular description of cellulose biosynthesis.” Annual review of biochemistry vol. 84 (2015): 895-921. https://doi.org/10.1146/annurev-biochem-060614-033930
2. 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
3. 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
## Co-culture
To achieve our goal of a stable co-culture that is viable to produce consistent mats with potentially homogoneus properties. We went through multiple engineering cycles to engineer Sticky yeast as well as attempting to develop a more standardised protocol to produce Bacterial cellulose mats, providing future iGEM teams with a potential baseline to produce, experiment and improve upon an BC mat.
<p>After our meeting with Tom Ellis we were faced with a dilemna that yeast tends to congregate at the bottom during standing cultivation. To solve this problem, we consulted with Prof Dr Daniel C. Ducat. During our meeting with himn he proposed several ideas including to engineer our yeast strain so that the outer cell wall has an increased affinity towards bacterial cellulose. Based on that, we did some research on yeast surface display systems and were also provided with a Saccharomyces Cerevisiae EBY100, an engineered strain that is commonly used for heterologous protein expression through an expression vector pYD1. Fortunately, the dye group already started working with cellulose binding domain </p>
<h2>Build</h2>
<p>We started, by amplifying the cellulose binding domain with the necessary overhangs then Enzyme digest for both the CBD and pYD1 expression vector. Afterwards we used T4 ligase to assemble our construct and after verification through cPCR transformed it succesfully in E. Coli.
We then verified the results through sequencing the CBD insert and finally transformed the expression vector with the CBD insert in *S. cerevisiae* EBY100. after 2 days initial colonies were visible and cPCR results show succesful transformation of S. Cerevisae</p>
<h2>Test</h2>
<p>To test Sticky yeast strain we attempted to perform a cell viability drop assay after induction of the expression vector. However due to time constraints we were not able to evaluate our results. In addition plans by using cellulose strips to compare the "stickiness" after induction in a sticky yeast culture were also considered but were feasibly not achieved</p>
<h2>Learn</h2>
<p>In the end we were not able to proof that Sticky yeast is a viable concept or not during our iGEM run.</p>
<summary><b>Stadardised BC mat protocol</b> Cycle 2</summary>
<h2>Design</h2>
<p>We initially designed all our in silico constructs for the expression of our chromoprotein constructs in E.coli with the terminator EF_TZ (BBa_J435371 )for our L1 constructs.</p>
<h2>Build</h2>
<p> To develop the Protocol we were researching different cultivation methods as well as investigating some of the initial BC mats cultivated by the other subgroups. by combining literature with our attempts we were able to write an initial experimental ressource</p>
<!-- if possible in time link to standardised BC mat protocol/-->
<h2>Test</h2>
<p>To verify the feasibility in producing any BC mat succesfully with the standardised protocol occured through a mini workshop to give members outside the Co-culture subgroup the chance to create a BC mat. The mats resulting from the test were later used in meetings with the fashion college, University of applied science in Niederrhein for further testing as well as dyeing experiments by the dye subgroup. However we were not able to meaningfully test if the mats were comparable due to time constraints. </p>
<h2>Learn</h2>
<p>During the proeject run we made several mistakes that resulted in Bacterial cellulose mats that were not really comparable and initial stages of the project we produced mats by inoculating rods. We also learned that the Cellulase amount could be reduced in half and still result in satisfactory results.</p>
</details>
<!--End Collaps section-->
## 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.
@@ -12,11 +12,17 @@ In the same test, we could prove that washing the bacterial cellulose with a 1%
In terms of drying conditions, drying in a drying cabinet at 50°C shows to increase the tensile strength and elasticity of BC mats compared to parchment drying.
