Part:BBa_K4119025
Pvgb-incert with 5'-UTR->Bs2->Cpa fdx terminator
Inserting exogenous 5'-UTR sequences downstream of the promoter vgb significantly enhances the expression effects of the promoter.
Result:
Inserting exogenous 5'-UTR sequences downstream of the promoter vgb significantly enhances the expression effects of the promoter.
Trough literature research and preliminary experiments, we found that the expression of the vgb promoter was not ideal, so we tried to enhance its ability to express proteins by the insert of a fragment of exogenous 5'-untranslated region(5'-UTR). In iGEM's official part registry catalog, we retrieved a translation enhanceing 5-UTR fragment(Part:BBa_K1758100) designed by team Bielefeld-CeBiTec in 2015, and we decided to insert this fragment downstream of the vgb promoter in the pre-constructed plasmid pMTL-vgb-bs2, hoping that the expression of the Bs2 fluorescent protein would improve. This sequence contains a 5'-UTR and a strong ribosomal binding site(RBS) from bacteriophage T7, and it's reported could greatly enhance translation of a following gene. The enhancing effect relies on the regulation of mRNA binding to and release of the ribosome S30 subunit. We have converted the construction and sequence analysis of this recombinant plasmid into E. coli CA434, but time remaining for the competition did not allow us to continue to transfer the recombined plasmid into C. tyrobutyricum by conjugation, so the detection of fluorescence intensity data was carried out in E. coli to verify the effectiveness of the improvements for the vgb promoter. By controlling aerobic and micro-aerobic culture conditions, we incubated E. coli bearing recombinant plasmid Pvgb-5'-UTR-bs2 together with Pvgb-bs2 as control, under those two conditions respectively, and sampled the culture solutions after logarithmic stages to detect fluorescence intensity after relevant treatment.
After collating and analyzing the fluorescence intensity data (Figure 8), it can be seen obviously that the improved promoter(Pvgb-5'-UTR) under aerobic conditions behaves similarly to the control group(Pvgb), while under the induction of microaerobic conditions, the expression effect of the improved promoter is 1.97-folds of the control group, which indicates that the promoter engineering strategy of inserting exogenous 5'-untranslated region(5'-UTR) does improve the expression of downstream proteins.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 396
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 396
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 396
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 396
- 1000COMPATIBLE WITH RFC[1000]
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