@@ -191,18 +191,18 @@ To achieve our goal to have controlled activation of the bacterial cellulose syn
<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>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 chloramphenicol and ampicillin 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><b>Figure 11</b>: DNA electrophoresis 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>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>
<p><b>Figure 11</b>: DNA electrophoresis gel picture 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>
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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. 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 came up with the idea of sticky yeast
To achieve our goal of a stable co-culture that is viable to produce consistent mats with potentially homogoneus properties. We went through multiple synbio and non synbio engineering cycles to improve cultivation methods and results.
<p>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>
<p>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 *Saccharomyces cerevisiae* EBY100, an engineered strain that is commonly used for yeast surface display purposes through an engineered expression vector pYD1 with multiple cloning sites to add a gene of interest. The gene of interest for our purposes is the cellulose binding domain. A binding protein with . Fortunately, the dye group already started working with cellulose binding domain sequence and already cloned it into an Level O construct. So we just needed to design primers to amplify the CBD with EcorI and XhoI overhangs to enable insertion at multiple cloning site in pYD1 later on.</p>
<p><b>Figure 1</b>: in silico construct of expression vector pYD1 with cellulose binding domain gene sequence insert.</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* DH5α.
We then verified the results through sequencing the pYD1 plasmid with 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>
<p>We started, by amplifying the CBD with EcorI and XhoI restriction site overhangs. Then to create sticky ends on both the backbone pYD1 and the CBD, enzyme digests has been done. Then followed a DNA purification through gel extraction and finally an assembly with T4 ligase. Afterwards we digested our backbone pYD1 and the CBD used T4 ligase to assemble our construct and finally transformed our construct in *E. Coli* DH5α and initial verification the presence of our insert through colony PCR.</p>
<p><b>Figure 2</b>: Colony PCR verification if CBD is in assembled construct.</p>
<p> We then verified the results through sequencing the pYD1 plasmid with CBD insert. After sequencing results verified that sequence and plasmid sequence are almost identical to in silico construct, we then transformed our construct in *S. cerevisiae* EBY100. After 2 days initial colonies were visible and cPCR results show succesful transformation of *S. Cerevisae* EBY100.</p>
<p><b>Figure 3</b>: *S. cerevisiae* EBY100 on drop out media -uracil -threonine agar plates after transformation with CD105 pYD1 CBD "Stcky yeast"(right). </p>
<p><b>Figure 4</b>: Colony PCR verification if plasmid with CBD insert is in *S. cerevisiae* EBY100.</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>
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<summary><b>Stadardised BC mat protocol</b> Cycle 2</summary>
<summary><b>Stadardised BC mat protocol</b> Cycle 1</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>
