Team:NAU-CHINA/Improve
Improved Parts
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Find Out MoreBackground
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.
We planned to insert an A/U-rich region in the upstream of the RBS of BBa_K528004
, and measure the florescence intensity of mRFP1. If the florescence intensity rises as the region inserted, the improvement shall be proved to be feasible.
Overview
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.
We planned to insert an A/U rich region in the upstream of the RBS of BBa_K528004, and measure the florescence intensity of mRFP1. If the florescence intensity rises as the region inserted, the improvement shall be proved to be feasible.
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 stabilizes the mRNA, making the mRNA to be functional in a longer period, promoting the protein synthesis.
Characterization
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 increases the secretion 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, the mRFP1 is synthesized more rapidly. As the part BBa_K528004 is a measurement device of RBS [measuring strength by measuring florescence intensity, then setting a standard values such as the florescence intensity of B0034, comparing the data and calculating the (florescence intensity sample) / (florescence intensity standard)], the measurement process could be much faster as the protein synthesis promoted. Besides, strength gaps among different RBS will also rise as the mRFP1 synthesized faster.