Difference between revisions of "Team:OUC-China/Improve"

Revision as of 03:55, 2 November 2017

Improve

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

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

@@ Line 130: / Line 130: @@
      </p>
      <p style="font-size: 20px">
-     	We selected J23108 from constitutive
+     	We selected BBa_J23108 from constitutive
-promoter family members and added the
+promoter family  and added a
-'UTR sequence thereafter. Fluorescent
+'UTR sequence that we choose from the paper mentioned above thereafter. Fluorescent
-protein is used to characterize whether the
+protein is used to characterize whether or not the
 'UTR has an enhanced effect on the
 promoter.
@@ Line 146: / Line 146: @@
      </p>
      <p style="font-size: 20px">
-     	We constructed two red fluorescent protein devices and constructed the loop by randomly
+     	We constructed a circuit devices with a commonly used red fluorescent protein and  randomly
-selecting the promoter and the terminator. The difference is that UTRrpsT is added to a loop and
+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 of 6h increased to 1.5 fold. This indicates that UTRrpsT has the capability
+fluorescence intensity at 6h increased to 1.5 fold. This indicates that UTRrpsT has the capability
-as a generic enhancement module.（Error bars represent standard deviation of three biological replicates.）
+to function as a generic enhancement module.（Error bars represent standard deviation of three biological replicates.）
 <h2 class="ouc-heading"><strong>Future work</strong></h2>
          <p style="font-size: 20px">
-         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.
+         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.
      </p>
      <h2 class="ouc-heading"><strong>Reference</strong></h2>