Team:OUC-China/RNA guardian/Design

We have added RBS to an mRNA 5’ Untranslated Region (UTR), single or poly. And we have designed a start codon and several stop codons in different reading frames. From a chemical perspective, the ribosome that is binding and dissociating will finally achieve a balance. We regard ribosome as a competitive inhibitor, inhibiting RNase E from being activated by binding on the 5’-end to some extents, as a result preventing mRNA degradation. In E.coli, many exonuclease such as polynucleotide phosphorylase (PNPase), RNase II, RNase R, and oligoribonuclease are also involved in the degradation progress. So, in the 3’ untranslated region, we designed a similar device.


(A)Genetic map of part K1059003


(B)Genetic map of part K1059004

Fig.1 Genetic map of part K1059003 and K1059004. (Here we named it “guardian”).

(A)We make part K1059003 a standard Biobrick by adding standard prefix and suffix. In that part, there’s a promoter(J23101) to start the transcript ,a RBS(B0035) to binding extra ribosome, a start codon to promote the binding efficiency of ribosome and several stop codon to prevent ribosome from going down. All of them are designed at 5’-UTR of GFP mRNA.

(B)At the 3’-UTR, we design part K1059004. In the part, there’re more than forty nucleotides between UAA and the extra RBS. The start and stop codon perform the same function as aprt K1059003.

Fig.2 A brief introduction to our expectation of RNA guardian.

From a chemical perspective, the ribosome is binding and dissociating from a mRNA and finally achieving a balance. At 5’-end UTR, We regard ribosome as a competitive inhibitor, inhibiting RNase E from being activated by binding on the 5’-end; at 3’-UTR, the ribosome performed the same function to another endonuclease.

We have constructed these following circuits. And to detect if the part works well, we use GFP as a reporter to reflect the translation efficiency. For experimental convenience, we choose GFP with a tag which can accelerate the degradation of GFP, So the fluorescence can reach a plateau quickly for easier measuring. To test the mRNA level, we extract the total RNA and proposed RT-PCR (reverse transcription-polymerase chain reaction).

Fig.3 Circuits constructed by OUC –iGEM.

In order to find which combination works well, we constructed these circuits and measured it by fluorescence and RT-PCR. All of the guardians are located in the untranslated region, 3’-end or 5’-end. Both of them consist of a RBS than can bind an extra ribosome, a start codon and several stop codons in different reading frames.

There may be a question troubling you. Will it lower the translational efficiency if the extra Ribosome interacts with the real ribosome? We have considered it. Between the two or several RBS, there are over forty nucleotides and that is considered enough to prevent two ribosomes from interfering with each other. Because of the several stop codons in different reading frames, the extra ribosome cannot go down the mRNA.