Team:NAU-CHINA/Basic Part

NAU-iGEM

Basic Part


Love something in one's life,
just as iGEMers love J04450. ——Guolong Shi

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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 OD600nm 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.


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