We achieved the production of bacterial cellulose through static culture. There is visible bacterial cellulose in the culture flask with HS media (Figure 1A). Quantitatively speaking, the wet weight of bacterial cellulose produced by G. xylinus in static culture is 17.847g, while the dry weight is 1.012g on average (Figure 1B).
As for the production of bacterial cellulose by Rotary Disc Reactor (RDR), because of the current pandemic of COVID-19, we haven’t performed experiments in this part. However, we have successfully constructed the hardware and debugged all the potential errors. We hope to implement the fermentation via RDR in the next iGEM competition.
Structural Protein Purification
- Figure 2. Electrophoresis for the genome PCR.
We tried to purify SpyCatcher003 and SpyTag003 based on two chassis organisms: and P. Pastoris and E. coli. However, the transformation and protein purification process for P. Pastoris are both troublesome. For the chemical transformation, the efficiency is not so ideal, which means that we need to repeat the transformation for many times in order to gain the correct clone. For the electroporation, although the efficiency is acceptable, the protocol for electroporation equipment is complex and also a little dangerous. Therefore, we finally achieved the successful transformation of P. Pastoris with genes responsible for SpyCatcher003 and SpyTag003 integrated with structural proteins according to genome PCR (Figure 2). However, we didn’t finish the subsequent protein purification process due to the limit of time.Two of the Pichia Pastoris strains GS115 were transformed with genes coding SpyCatcher003 with CBM and SpyTag003 with Scl2, respectively, under the control of AOX1 promoter. The marker in the electrophoresis is composed by DNA bands with length 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, 3000bp, 5000bp, and 8000bp. The lengths of target fragments are both about 4000bp, so the electrophoresis report shows that transformations are successful.
- Figure 3. SDS-PAGE result of target proteins.
As for the protein purification from E. coli. The SDS-PAGE result showed that the proteins is not successfully synthesized. Instead, they might be digested to become several fragments of polypeptides with different length (Figure 3). The possible reason for the failure of protein purification in E. coli might be attributed to the length of CDS. The DNA length of SpyCatcher003 with cellulose binding module and SpyTag003 with structural proteins are both about 4kb, which might be exceedingly long beyond the endurance limit of E. coli. Another reason is that protease inhibitor was not adopted in our protein purification protocol, which might cause the digestion of our target protein.
Protein bands with different length overlapped with each other to form a continuous pattern of the lane as a whole. The pattern suggests digestions might occur, cutting target proteins to several fragments of polypeptide with different length.
Our project was negatively affected by COVID-19 pandemic. However, our team members for this year and the prospective team leaders for iGEM 2021 BNDS_China all feel that this project is worthy to be continued. Therefore, we choose to be a Two-Phase project for the next year iGEM competition. Our project next year would be based on the theoretical design and modeling prediction done in iGEM 2020, moving forward to experiment implementation if the pandemic were relieved.
For the production of bacterial cellulose via RDR, we plan to manufacture the hardware in large scale so that we could have multiple equipment working at the same time to achieve the mass production of bacterial cellulose. Moreover, we decided to adopt Yamanaka Medium Ethanol (YME), which is a brand-new culture media for the production of bacterial cellulose with more nutrition, in order to enhance the productivity. In addition, the strain for cellulose production could be various. We plan to make a comparison between G. xylinus, G. hansenii, and several other kinds of cellulose producing strain in order to find the optimized one.
For the protein purification, we decided to make thorough investigation regarding the fermentation conditions for P. Pastoris. For example, the concentration of methanol, the inducer for the protein expression in P. Pastoris, is debatable according to academic literature report. Moreover, the different integration site of linearized expression cassette in P. Pastoris genome would influence the utilization efficiency of methanol, producing the Mut+ type (normal methanol utilization) and MutS type (slow methanol utilization). The choice of different integration sites is also debatable, so we decide to try both and adopt the better one. As for the protein expression in E. coli, we plan to verify the endurance limit of this chassis and add protease inhibitor to avoid digestion of target proteins. If the protein expression in E. coli still didn’t work, we would focus on P. Pastoris only.
In conclusion, we are looking forward to further continuing this project in iGEM 2021 as a Two-Phase project. With the basis of theoretical design and analysis, we believe that our experiment implementation would flow well next year.