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© Research
Publication : ACS synthetic biology

Tuning of Gene Expression in Clostridium phytofermentans Using Synthetic Promoters and CRISPRi.

Scientific Fields
Diseases
Organisms
Applications
Technique

Published in ACS synthetic biology - 16 Dec 2022

Rostain W, Zaplana T, Boutard M, Baum C, Tabuteau S, Sanitha M, Ramya M, Guss A, Ettwiller L, Tolonen AC

Link to Pubmed [PMID] – 36427328

Link to DOI – 10.1021/acssynbio.2c00385

ACS Synth Biol 2022 Dec; 11(12): 4077-4088

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.