This year, our team improved the promoter BBa_J23018 in a novel way. The newly constructed promoter is BBa_K2314212, in which we add a UTRrpsT sequence and enhanced its strength. Considering relative researches and the result of our experiments, we have the reason to believe that UTRrpsT has the potential to enhance the promoter as a universal enhancement module.

Inspiration

As known to all, 5'UTR will be transcribed and can be used as a regulatory element to adjust the translation process. Therefore, it is believed 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 and helped improve the expression of target protein. Thinking about our need to express target gene in our project, we decided to edit an existing promoter in Part in this way. Hopefully, the strength will be enhanced. Since the promoter we picked was not operated this way before, we were not sure about its result at first, so we consulted the auther of our basic paper. Luckily,the idea also received the author's support.[1]

Design

We selected BBa_J23108 from constitutive promoter family and added a 5'UTR sequence that we choose from the paper mentioned above thereafter. Fluorescent protein is used to characterize whether or not the 5'UTR has an enhanced effect on the promoter.

Proof of concept

We constructed a circuit devices with a commonly used red fluorescent protein and randomly selected 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 at 6h increased to 1.5 fold. This indicates that UTRrpsT has the capability to function as a generic enhancement module.（Error bars represent standard deviation of three biological replicates.）

Future work

According to the analysis of data, we may draw a conclusion that the part can impove the expression of our target protein (mRFP). In the future, we can change different reporters to detect whether different reporters may have the same result---that their expression will all be improve. If so, we can make sure that the part is universal for improving all proteins and 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 might offer help to other teams.

Reference