Team:OUC-China/Improve

Improve

With a new entry point, our team enhanced the strength of the promoter BBa_J23018. The newly constructed promoter is BBa_K2314212, in which we add a UTRrpsT sequence. And we have reason to believe that UTRrpsT has the potential to enhance the promoter as a universal enhancement module.

Inspiration

5'UTR will be transcribed and can be used as a regulatory element to adjust the translation process. Thus, there is reason to believe that a delicate structure of the 5'UTR has the potential as a universal enhancement module. We see that Zhou et al. Screened some 5'UTR sequences from E. coli, which helped to improve protein content. Thus, we are prepared to consider this part as an enhancement module, the idea also received the author's support.[1]

Design

We selected J23108 from constitutive promoter family members and added the 5'UTR sequence thereafter. Fluorescent protein is used to characterize whether the 5'UTR has an enhanced effect on the promoter.

Design

We selected J23108 from constitutive promoter family members and added the 5'UTR sequence thereafter. Fluorescent protein is used to characterize whether the 5'UTR has an enhanced effect on the promoter.

Proof of concept

We constructed two red fluorescent protein devices and constructed the loop by randomly selecting the promoter and the terminator. The difference is that UTRrpsT is added to a loop and the fluorescence intensity is measured using a microplate reader and plotted. We found that the fluorescence intensity of 6h increased by 1.5 fold. This indicates that UTRrpsT has the capability as a generic enhancement module.

Future work

According to analysis of data, we may draw a conclusion the part can impove the expression of our target protein (mRFP). In the future, we can change different reporters to wonder whether different reporters can have the same result that their expression are all improved, if so we can make sure that the part is universal for improving all proteins. Therefore we can utilize this provement to increase the expression of mSA, which improve the probability of binding to yeast successfully and strengthen the binding. Further we offer the part to other teams.

Reference

[1]Zhou, Shenghu, et al. "Obtaining a Panel of Cascade Promoter-5′-UTR Complexesin Escherichia coli." Acs Synthetic Biology 6.6(2017)