Team:NAU-CHINA/Basic Part

NAU-iGEM

Abstract

This year, NAU-CHINA build 14 basic parts, including 9 single anti-Plasmodium peptide, hasA hemophore, TEV cleavage site, TEV protease, the fusion anti-plasmodium protein coding sequence and A/U-rich 5’-UTR.

Our favorite basic part is A/U-rich 5’-UTR (BBa_K3139001).
We aim to add a new tool to improve the efficiency of protein syhthesis.

Background

This sequence can be added at the 5'-terminal of the RBS (between the promoter and RBS). In E.coli hosts, the ribosomal protein S1 could bind with the mRNA region which is encoded by this part, the stability of the mRNA and the possibility of ribosome binding shall rise at the same time. As a consequence, the expression of the gene which was added with this part can be obviously improved. Expression of high-abundance proteins, like phage coat proteins, ATP synthase proteins, translational factors, or nucleoid proteins, is accompanied by an A/U-rich region upstream of the translation initiation codon and the ribosome-binding site, and this region acts as an "enhancer" by attracting ribosomal protein S1.

Fig.1 The mechanism of A/U-rich 5'-UTR to promote protein synthesis.
(1) The A/U-rich 5'-UTR induces the ribosomal protein S1 to bind to the RBS, promoting the translation process and accelerating the protein synthesis.
(2) Induced ribosomes bind to the mRNA more frequently which stablizes the mRNA, making the mRNA to be functional in a longer period, promoting the protein synthesis.

This part can completely work only with interaction between this part and ribosomal protein S1. If the ribosomal protein S1 gene get mutated, not only the function shall be inhibited, but also the viability shall be badly damaged. So this part should be effective in most strains of E. coli and other species.

We attempt to insert this sequence into BBa_K528004 (between BBa_J23100 and BBa_B0032)， thus improve the characterize efficiency of protein-coding parts. This improved part works as expectation.

Though the function of this part was proved basically, more sequences with different UTR, promoters, RBS, and CDS are needed for further exploration.

Experiment

We constructed two plasmids by using homologous recombination, which with UTR sequence inserted and without UTR sequence inserted. Both recombinant plasmids were transformed into E.coli DH5α competent cells and grown overnight. Single colonies were used to inoculate in 50ml LB medium containing chloramphenicol and cultured for 16 hours. Taking 20μL bacterial fluids and putting it into 1.2 ml LB medium. Taking out the bacterial fluids after 0, 4, 8, 12, 16 hours, which consists of 3 biological repeats. We separately removed 100μL fluids to the plate, then centrifuged the remaining fluids, removed 100μL supernatant to the plate, and removed the rest of the fluid. Cells were resuspended with 320μL 1 x PBS and removed 100μL fluids to the plate. Using the plate reader to measure the fluorescence and OD600nm of the sample.

Result

Fig.2 Data obtained by plate reader
(A)Measurement of OD_600nm of DH5α.
(B)Measurement of RFP fluorescence intensity at 611nm (excitation at 584nm) of DH5α.

The figure shows that the distinction among OD600nm data were not clear between 2 treatments, which means that we can still compare the fluorescence intensity as they probably were in the same growth phase.
The sample with UTR sequence increase the fluorescence intensity to approximately 2-fold, which means RFP characterize level has significant improvement. It shows that UTR greatly improves the efficiency of protein characterization.

In conclusion, UTR coding sequence works well as expected, so we present A/U-rich 5’-UTR (BBa_K3139001) as our best basic part.

Here are all the basic parts:

Part Number	Name	Type
BBa_K3139000	Anti-plasmodium peptide MeuTXKβ1	Coding
BBa_K3139001	A/U-rich 5’-UTR	other
BBa_K3139002	Anti-plasmodium peptide drosomycin	Coding
BBa_K3139003	Anti-plasmodium peptide Gomesin	Coding
BBa_K3139004	Anti-plasmodium peptide Shiva-3	Coding
BBa_K3139005	Anti-plasmodium peptide MP2	Coding
BBa_K3139006	Anti-plasmodium peptide AdDLP	Coding
BBa_K3139007	Anti-plasmodium peptide Vida3	Coding
BBa_K3139008	Anti-plasmodium peptide SM1	Coding
BBa_K3139009	Anti-plasmodium peptide Thiostrepton	Coding
BBa_K3139010	hasA hemophore coding sequence	Coding
BBa_K3139011	TEV protease recognition and cleavage site	Coding
BBa_K3139012	TEVpS	Coding
BBa_K3139013	Anti-plasmodium fusion protein	Coding

Reference

[1]Khosa S , Scholz R , Schwarz C , et al. An A/U-rich enhancer region is required for high-level protein secretion through the HlyA Type I secretion system[J]. Applied and Environmental Microbiology, 2017:AEM.01163-17.