5'-UTR Sequence Mining with Consistent Regulation in Escherichia coli in Vitro and in Vivo
Objective:The standardization and modularization of genetic elements provide a reference for the transformation and design of increasingly complex genetic systems.By exploring the consistency of the 5'-untranslated region(5'-UTR)in regulating translation initiation in E.coli in vivo and in vitro,the universal 5'-UTR sequence is mined to add genetic elements with fine characteristics to modular engineering.Methods:A random library of 5'-UTR containing 25 nt was constructed and transferred into E.coli,and the relative activity in vivo was quantified by measuring the fluorescence value(Flu)and OD600 of the sample.The protein expression in vitro was performed using the cell extract of E.coli BL21 Star(DE3),and the relative activity in vitro was quantified by measuring the Flu of the sample.The sequence characteristics of the library were analyzed according to the base distribution and the minimum free energy of the 5'-UTR.The real activity values in vitro and in vivo were correlated and the Pearson correlation coefficient was calculated.Results:554 5'-UTR variants were successfully constructed and showed a moderate correlation in the in vitro and in vivo protein expression system(Pearson'r=0.64).Among them,the 5'-UTR sequence with high relative activity value is rich in AG,and its MFE is large in vivo and in vitro,but the hybridization energy MFE with 16S rRNA is small.The size of the library would have some fluctuation on the correlation analysis in vitro and in vivo,but it was weak;Finally,it was found that the 5'-UTR sequences in the high activity interval were highly correlated in vivo and in vitro,and three 5'-UTR sequences were selected from the library to be used in the fields of metabolic engineering and synthetic biology.Conclusion:The moderate correlation between in vitro and in vivo protein expression promotes the expansion and application of rapid in vitro characterization in the study of in vivo biosynthesis,and the screened universal 5'-UTRs can provide a choice for the genetic design of the genetic circuit.
5'-UTRE.coliGene expression regulationCell-free protein expressionSynthetic biology
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