CONTRIBUTION
BUCT worked with JLU-China, Hainan_China, BNU-China, DKU_China and other teams to create a beginner's manual. This manual is used to help new iGEMers integrate into iGEM. There are new iGEMers who join the iGEM team every year, but a lot of the experience needs to be gained autonomously.
As a multiple iGEM team, we still face many of the same problems that new teams face every year. And every time we learn, we get experience from the previous year's senior players. This year, in the process, we started thinking about how to make this learning process easier. Therefore, we participated in the creation of the manual jointly initiated by Hainan_China and JLU-China.
We hope this experience book will be useful for future teams. It would be great to bring confidence and experience to every new iGEMer who needs to learn. This Handbook can facilitate new people's understanding of iGEM events.
BUCT adds a new document to the registry page for BBa_K1060000 and adds new information.
We learned a new aroG sequence from the literature. Through homology modeling, we compared the structure of BBa_K4146502 and BBa_K1060000, and analyzed the reason why the phenylalanine (Phe) inhibition of our aroG mutant sequence was weakened. We used this gene for the synthesis of 2-phenylethyl alcohol, and finally edited the yield of this synthetic use in registry page.
Two interrelated pathways of conformational change in AroG transmit inhibitory signals from the Phe binding site to the active site of DAHP. This involves changes in the contact between subunits. Generally, there are two ways to weaken Phe and inhibit AroG. One is to inhibit the binding of Phe to AroG, and the other is to affect the conformation of Phe after binding to AroG.
The mechanism by which Phe inhibits AroG activity is very complex. The new AroG binds to Phe and the whole molecule undergoes a massive conformational change. It has been shown that our mutant aroGfbr can relieve the inhibition of Phe analogue 4-fluoro-DL-phenylalanine (p-EP). p-EP is a stronger inhibitor of aroG than Phe, and Escherichia coli cannot grow when aroG is inhibited by p-EP. We visualized this part of the conformational change through homology modeling, hoping to provide more references for future teams in this way.
The method of modeling can predict the actual growth and yield of E. coli in reality. During the modeling, we did the detailed calculation, and these calculation steps are preserved. In combination with growth, competitive inhibition and yield, our calculations were mainly aimed at obtaining a quorum sensing promoter suitable for CONTROLLING the EXPRESSION of toxic proteins and predicting the YIELD of important substances. (More details see our model )
If a team has similar needs in the future, they can learn from these computational steps. In our analysis, the new AroG could resist the inhibitory effect of Phe because the new conformation caused some obstacles to the combination of Phe and AroG.