在大肠杆菌体内外调控一致的5′-UTR序列挖掘  

5'-UTR Sequence Mining with Consistent Regulation in Escherichia coli in Vitro and in Vivo

作　　者：李佩贤 李娇娇 刘倩[1,2] 张婕[1,2] 齐浩[1,2] LI Peixian;LI Jiaojiao;LIU Qian;ZHANG Jie;QI Hao(School of Chemical Engineering and Technology,Tianjin University,Tianjin 300350,China;Key Laboratory of Systems Bioengineering of Ministry of Education,Tianjin University,Tianjin 300350,China)

机构地区：[1]天津大学化工学院,天津300350 [2]天津大学系统生物工程教育部重点实验室,天津300350

出　　处：《中国生物工程杂志》2024年第5期20-31,共12页China Biotechnology

基　　金：国家重点研发计划(2019YFA090007)资助项目。

摘　　要：基因元件的标准化和模块化为日益复杂的遗传系统改造设计提供了参考。目的:通过探究5′-非翻译区(5′-untranslated region,5′-UTR)在大肠杆菌体内外对翻译起始调控的一致性,挖掘通用型5′-UTR序列为模块化工程增添具有良好特征的遗传元件。方法:构建包含25 nt的5′-UTR随机文库化转至大肠杆菌中,通过测量样品荧光值(Flu)和OD600来量化其体内的相对活性值;利用大肠杆菌BL21 Star(DE3)细胞抽提物进行体外蛋白质表达,通过测定样品Flu来量化体外的相对活性值;依据5′-UTR的碱基分布、最小自由能等对文库序列特征进行分析;将得到的体内外真实活性值进行相关性分析并计算皮尔逊相关系数。结果:成功构建的554个5′-UTR变体文库在体内外蛋白质表达系统中表现出中等相关性(Pearson’r=0.64);其中,相对活性值高的5′-UTR序列富含AG,并且其在体内外均呈现自身最小自由能较大,而与16S rRNA杂交的自由能较小;文库数据量大小使体内外相关性分析产生一定的波动性但较为微弱;最后,发现高活性区间内的5′-UTR序列在体内外相关性较高,并从文库中优选出3条作为通用型5′-UTR应用于代谢工程与合成生物学领域。结论:体内外蛋白质表达的中等相关性推动了体外快速表征在研究体内生物合成中的拓展与应用,筛选出的通用型5′-UTR为基因线路的遗传设计提供了选择。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:5545'-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 ci

关 键 词：5′-非翻译区 大肠杆菌 基因表达调控 无细胞蛋白表达 合成生物学 

分 类 号：Q812[生物学—生物工程]