Creating a Gene Expression Cassette for the Cyanobacterium Synechococcus sp. PCC 7002 - And Using it to Replace the Endogenous Hydrogenase
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Eukaryotic algae and cyanobacteria are a promising source of future biofuels, having many important advantages over 1st and 2nd generation biofuels. Synechococcus sp. PCC7002 is the fastest growing oxygen producing photoautotroph known at present, and is capable of surviving a large wide of environments. As such, it is a very interesting organism for the production of biofuels. This is the first work that involves Synechococcus sp. PCC7002 at NTNU. In order to develop this organism into a hydrogen-producing organism, the native and inefficient [NiFe]-hydrogenase should be deleted and replaced by an energetically poised efficient [FeFe]-hydrogenase. A codon-optimized gene for expressing a [FeFe]-hydrogenase was designed. A plasmid was designed to allow for easy transformation and expression of exogenous genes, including the codon-optimized [FeFe]-hydrogenase, in Synechococcus. It contained a high expression promoter, capable of driving expression of proteins in a related cyanobacteria to 15% of the cell's dry weight. A temporary marker gene, mRFP1, was used to verify transformation. However, due to time-constraints, this overexpression system was not evaluated in Synechococcus. In addition, a plasmid was constructed that could delete the native [NiFe]-hydrogenase in Synechococcus. Unfortunately, Synechococcus could not be succesfully transformed with this deletion plasmid. Further, using the gene expression plasmid, a different hydrogenase could be expressed in this strain. Modifications to the Synechococcus transformation protocol are discussed, to help future researchers at NTNU.