Tuning of Gene Expression in Clostridium phytofermentans Using Synthetic Promoters and CRISPRi,ACS Synthetic Biology

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Tuning of Gene Expression in Clostridium phytofermentans Using Synthetic Promoters and CRISPRi
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2022-11-25 , DOI: 10.1021/acssynbio.2c00385
William Rostain ₁ , Tom Zaplana ₁ , Magali Boutard ₁ , Chloé Baum _{1,

2} , Sibylle Tabuteau ₁ , Mary Sanitha ₃ , Mohandass Ramya ₃ , Adam Guss ₄ , Laurence Ettwiller ₂ , Andrew C Tolonen ₁

Affiliation

Control of gene expression is fundamental to cell engineering. Here we demonstrate a set of approaches to tune gene expression in Clostridia using the model Clostridium phytofermentans. Initially, we develop a simple benchtop electroporation method that we use to identify a set of replicating plasmids and resistance markers that can be cotransformed into C. phytofermentans. We define a series of promoters spanning a >100-fold expression range by testing a promoter library driving the expression of a luminescent reporter. By insertion of tet operator sites upstream of the reporter, its expression can be quantitatively altered using the Tet repressor and anhydrotetracycline (aTc). We integrate these methods into an aTc-regulated dCas12a system with which we show in vivo CRISPRi-mediated repression of reporter and fermentation genes in C. phytofermentans. Together, these approaches advance genetic transformation and experimental control of gene expression in Clostridia.

中文翻译：

使用合成启动子和 CRISPRi 调节 Clostridium phytofermentans 中的基因表达

基因表达的控制是细胞工程的基础。在这里，我们展示了一系列使用模型Clostridium phytofermentans来调节梭状芽胞杆菌中基因表达的方法。最初，我们开发了一种简单的台式电穿孔方法，用于鉴定一组可共转化至C. phytofermentans 的复制质粒和抗性标记。通过测试驱动发光报告基因表达的启动子库，我们定义了一系列跨越> 100倍表达范围的启动子。通过在报告基因上游插入tet操纵位点，可以使用 Tet 阻遏物和脱水四环素 (aTc) 定量改变其表达。我们将这些方法整合到 aTc 调节的 dCas12a 系统中，通过该系统，我们在C. phytofermentans中展示了CRISPRi 介导的报告基因和发酵基因的体内抑制。这些方法共同促进了梭状芽胞杆菌基因转化和基因表达的实验控制。

更新日期：2022-11-25

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