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CRISPRi–sRNA: Transcriptional–Translational Regulation of Extracellular Electron Transfer in Shewanella oneidensis
journal contribution
posted on 2017-06-15, 12:52 authored by Yingxiu Cao, Xiaofei Li, Feng Li, Hao SongExtracellular
electron transfer (EET) in Shewanella
oneidensis MR-1, which is one of the most well-studied
exoelectrogens, underlies many microbial electrocatalysis processes,
including microbial fuel cells, microbial electrolysis cells, and
microbial electrosynthesis. However, regulating the efficiency of
EET remains challenging due to the lack of efficient genome regulation
tools that regulate gene expression levels in S. oneidensis. Here, we systematically established a transcriptional regulation
technology, i.e., clustered regularly interspaced short palindromic
repeats interference (CRISPRi), in S. oneidensis MR-1 using green fluorescent protein (GFP) as a reporter. We used
this CRISPRi technology to repress the expression levels of target
genes, individually and in combination, in the EET pathways (e.g.,
the MtrCAB pathway and genes affecting the formation of electroactive
biofilms in S. oneidensis), which in
turn enabled the efficient regulation of EET efficiency. We then established
a translational regulation technology, i.e., Hfq-dependent small regulatory
RNA (sRNA), in S. oneidensis by repressing
the GFP reporter and mtrA, which is a critical gene
in the EET pathways in S. oneidensis. To achieve coordinated transcriptional and translational regulation
at the genomic level, the CRISPRi and Hfq-dependent sRNA systems were
incorporated into a single plasmid harbored in a recombinant S. oneidensis strain, which enabled an even higher
efficiency of mtrA gene repression in the EET pathways
than that achieved by the CRISPRi and Hfq-dependent sRNA system alone,
as exhibited by the reduced electricity output. Overall, we developed
a combined CRISPRi–sRNA method that enabled the synergistic
transcriptional and translational regulation of target genes in S. oneidensis. This technology involving CRISPRi–sRNA
transcriptional–translational regulation of gene expression
at the genomic level could be applied to other microorganisms.
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Keywords
genome regulation toolsEET pathwaystranscriptional regulation technologytarget genestranslational regulation technologyMRgene expression levelsgenomic levelmtrA gene repressionHfq-dependent sRNA systemsHfq-dependent sRNA systemShewanella oneidensis Extracellular electron transferCRISPRiExtracellular Electron TransferGFPtranslational regulation
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