CRISPR/Cas9n knock-out plasmid construction and transformation into Clostridium autoethanogenum for deletion of a formate dehydrogenase gene
Date
2023
Authors
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Publisher
Tartu Ülikool
Abstract
Gas fermentation using acetogen bacteria is an attractive technology for tackling the
problem of the accumulation of solid waste and greenhouse gases while contributing
towards a circular economy through sustainable production of chemicals and fuels.
Acetogens are the preferred biocatalysts for gas fermentation as they can use gases such as
carbon monoxide (CO) and carbon dioxide (CO2) as their sole carbon source. They fix
carbon oxides via the Wood-Ljungdahl pathway (WLP), but specific functionalities of
several key enzymes in the WLP are unclear. This thesis constructed a CRISPR/Cas9
nickase (Cas9n) plasmid for targeted deletion of the formate dehydrogenase gene fdhA in
the model-acetogen Clostridium autoethanogenum that can catalyse the reduction of CO2
to formate in the WLP. The fdhA knock-out plasmid was constructed using PCR-based
cloning methods and InFusion cloning. The latter was also used to insert homology arms
flanking the fdhA gene into the knock-out plasmid that are needed to facilitate gene
deletion via homologous recombination in C. autoethanogenum. The plasmid was
successfully transformed into C. autoethanogenum but fdhA deletion-bearing cells were
not identified. Future work includes sub-culturing and plating the transformed cells to
isolate fdhA knock-out cells, plasmid curing, and growth characterisation of the fdhA
knock-out strain. This thesis provides both an intermediate strain for further study and
valuable information for optimisation of future genetic engineering efforts.
Description
Keywords
clostridium autoethanogenum, gas fermentation, genetic engineering, CRISPR/Cas9n