Due to the complexity of biological systems, engineering it in any way is a hard, and often repetitive process of trial and error. In our project, we went through multiple cycles of design, engineering, and testing. From these steps, we obtained information, that went into rethinking and redesign of the project. On this page, we attempt to demonstrate our engineering cycle and out journey to success.
Summary of our troubleshooting and optimization of the following parts to transform into E. coli.
After months debate about a problem to solve, we identified the need for an easily obtainable, in vivo degradable and biocompatible material to use in medical applications. During brainstorming sessions, different options were presented as potential candidates from reviewing of pre-existing literature. From a paper published by Yadav et al. (2010), we were inspired by their idea to generate a material by fusing cellulose monomers with chitin elements, using the endogenous machinery of Komagataeibacter for cellulose production. We thought of improving this implementation by circumventing the need for amine sugars as a substrate thereby expanding upon the original work. Through the KEGG database, and literature three additional genes were selected in addition to two genes from the original publication. The design was changed multiple times throughout the project in response to problems in implementation or negative results. A more detailed explanation of this can be found in the Experiments page.
After the completion of the experimental design, we moved forward in the lab to bring the idea into reality. Initially we aimed at amplifying 3 genes from S. cerevisiae genomic DNA (gDNA) with the help of PCR. The primers were designed in away to allow for generation of parts that can be ligated together after restriction enzyme digestion. In addition to the above cloning approach we ordered parts from IDTDNA to be synthesized and sent to us. Our initial attempts were aiming to clone the necessary parts into certain cloning vector (pUC19) and the whole construct being under the control of the strong and constitutive promoter Bla. The attempts at amplifying the 3 individual genes from S. cerevisiae gDNA were successful. However, our ligation attempts either with restriction enzyme digestion approach or with isothermal assembly attempts were not successful. Eventually we opted for new designs utilizing a different promoter (J23110 from the iGEM Anderson promoter list) which led to the same results. Eventually we generated new designs, aiming at cloning the 3 necessary genes, plus one more ( GFA1? Convert fructose-6P to glucosamine-6P) and the target was a different expression vector (pASK-IBA3) under the control of the same constitutive promoter (J23110). After this attempt not yielding the desired results we decided to troubleshoot the cloning protocol, by isolating one gene (UAP1) and clone it directly in the pASK-IBA3 to verify that our cloning strategy is sound and at the same time demonstrate that the part in question, expresses properly using the pTet promoter from the pASKIBA3 vector which is an inducible promoter (turns on upon addition of anhydrotetracycline). Finally we successfully amplified UAP1 from yeast gDNA, cloned it into pASK-IBA3 and transformed it into E.coli where we attempted to express the protein. During a fast protein expression protocol we obtained crude protein lysate that we analysed with the help of SDS-PAGE and confirmed the expression of the gene of interest upon induction. At last we wanted to demonstrate the functionality of the protein expressed by using the crude cell lysate in a phosphatase assay, however we did not manage to finalize this aspect, although we have successfully used a colorimetric assay to detect phosphatase activity. This was necessary due to growth in recombinant organisms of the entire construct, which made us suspect that the changes were toxic to the bacteria. We went through two different designs, one using the original plasmid and process, mirroring the original publications method, and another attempt using a process recommended to us. We ended up transforming the single gene into our intermediate host, Escherichia coli, and proceeded to test this to validate insert and functional protein activity. More details on this process can be found in the Notebook and Experiments page.
As part of the troubleshooting, we had to test the intermediate host thoroughly. Because of this, we tested both for proof of recombinant DNA insert with PCR amplification and digestion enzymes, and protein expression using SDS-PAGE and colorimetric assays. From this, we did not see the presence of recombinant DNA, but did see an increase in protein expression of the expected protein by size, and an increase in protein expression with increased metabolism of phosphates, the function of the protein encoded by recombinant DNA that we attempted to insert. With sequencing, we finally found that the plasmid did not contain our target DNA. A more detailed explanation of this process can be found in the Notebook, Experiments and Results.
As explained above, this did not work as expected with all our experiments up-to this point. From this sets of trial and errors, we gained valuable knowledge. This tells us that the way we constructed and attempted to transform our bacteria was less than ideal and could even be toxic to our cell. And made us rethink the entire process; thus, a new engineering cycle began.
