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Modulating Heterologous Gene Expression with Portable mRNA-Stabilizing 5′-UTR Sequences
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2018-07-31 00:00:00 , DOI: 10.1021/acssynbio.8b00191 Sandra C. Viegas 1 , Patrícia Apura 1 , Esteban Martínez-García 2 , Víctor de Lorenzo 2 , Cecília M. Arraiano 1
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2018-07-31 00:00:00 , DOI: 10.1021/acssynbio.8b00191 Sandra C. Viegas 1 , Patrícia Apura 1 , Esteban Martínez-García 2 , Víctor de Lorenzo 2 , Cecília M. Arraiano 1
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RNA half-lives are frequently perceived as depending on too many variables, and transcript stability is generally missed as a checkpoint amenable to manipulation in synthetic designs. In this work, the contribution of mRNA stability to heterologous protein production levels in E. coli has been inspected. To this end, we capitalized on the wealth of information available on intrinsic mRNA stability determinants, four of which were formatted as portable modules consisting of 5′-untranslated regions (UTRs). The cognate DNA sequences were then assembled in a genetic frame in which mRNA stability endowed by the UTRs was the only variable to run expression of sfGFP. Reporter output and Northern blot-based measurements of absolute mRNA half-lives revealed that such UTRs were found to keep intact their ability to modulate transcript stability when excised from their natural context and placed as the upstream region of the reporter gene. By keeping transcription fixed and combining different UTRs with a constant ribosomal binding site, we showed that mRNA decay can be made the limiting constituent of the overall gene expression flow. Moreover, the data indicated that manipulating mRNA stability had little effect on expression noise in the corresponding population. Finally, augmented heterologous expression brought about by mRNA stability did not make cells more vulnerable to resource-consuming stresses. The tangible result of this work was a collection of well-characterized mRNA-stabilizing sequences that can be composed along with other expression signals in any construct following the assembly rules of the Standard European Vector Architecture (SEVA) format.
中文翻译：
用稳定的mRNA稳定的5'-UTR序列调节异源基因表达。
RNA半衰期通常被认为取决于太多变量，而转录物稳定性通常被遗忘为适合合成设计操作的检查点。在这项工作中，mRNA稳定性对大肠杆菌中异源蛋白质生产水平的贡献已经检查过了。为此，我们利用了有关内在mRNA稳定性决定因素的大量信息，其中四个被格式化为由5'-非翻译区（UTR）组成的便携式模块。然后将相关的DNA序列组装在一个遗传框架中，在该遗传框架中，UTR赋予的mRNA稳定性是运行sfGFP的唯一变量。报告者的输出和基于RNA的绝对mRNA半衰期的测量结果表明，从自然环境中切除并放置为报告基因的上游区域后，发现此类UTR保持了完整的调节转录稳定性的能力。通过保持转录固定并结合具有恒定核糖体结合位点的不同UTR，我们证明了mRNA衰变可以成为整个基因表达流程的限制性组成部分。而且，数据表明操纵mRNA的稳定性对相应群体中的表达噪声影响很小。最后，由mRNA稳定性带来的增强的异源表达并未使细胞更容易受到资源消耗的压力的影响。这项工作的切实结果是，收集了一系列充分表征的mRNA稳定序列，这些序列可以与其他表达信号一起，按照标准欧洲矢量体系结构（SEVA）格式的装配规则在任何结构中组成。
更新日期：2018-07-31
中文翻译：
用稳定的mRNA稳定的5'-UTR序列调节异源基因表达。
RNA半衰期通常被认为取决于太多变量，而转录物稳定性通常被遗忘为适合合成设计操作的检查点。在这项工作中，mRNA稳定性对大肠杆菌中异源蛋白质生产水平的贡献已经检查过了。为此，我们利用了有关内在mRNA稳定性决定因素的大量信息，其中四个被格式化为由5'-非翻译区（UTR）组成的便携式模块。然后将相关的DNA序列组装在一个遗传框架中，在该遗传框架中，UTR赋予的mRNA稳定性是运行sfGFP的唯一变量。报告者的输出和基于RNA的绝对mRNA半衰期的测量结果表明，从自然环境中切除并放置为报告基因的上游区域后，发现此类UTR保持了完整的调节转录稳定性的能力。通过保持转录固定并结合具有恒定核糖体结合位点的不同UTR，我们证明了mRNA衰变可以成为整个基因表达流程的限制性组成部分。而且，数据表明操纵mRNA的稳定性对相应群体中的表达噪声影响很小。最后，由mRNA稳定性带来的增强的异源表达并未使细胞更容易受到资源消耗的压力的影响。这项工作的切实结果是，收集了一系列充分表征的mRNA稳定序列，这些序列可以与其他表达信号一起，按照标准欧洲矢量体系结构（SEVA）格式的装配规则在任何结构中组成。
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