Methods for Fully Segregating Recombinant Marine Cyanobacteria

A method for isolating recombinant Synechococcus sp. strain PCC 7002 containing a recombinant form of a high copy number endogenous plasmid and lacking the wild-type form of said high copy number endogenous plasmid, said recombinant form of a high copy number endogenous plasmid comprising a wild-type high copy number endogenous plasmid backbone and an exogenous polynucleotide sequence insert, said exogenous polynucleotide sequence insert comprising a selective marker, said method comprising:

• a) transforming Synechococcus sp. strain PCC 7002 comprising said wild-type high copy number endogenous plasmid with an exogenous polynucleotide,
• b) culturing said transformed Synechococcus sp. strain PCC 7002 in media comprising a selective agent,
• c) culturing said cultured transformed Synechococcus sp. strain PCC 7002 from step b) on low salt media comprising concentrations of a selective agent,
• d) selecting individual colonies from a medium of step c),
• e) culturing said selected individual colonies from step d) on said medium of step d),
• f) selecting colonies from said medium of step e),
• g) culturing said selected colonies from step f) on a high salt medium,
• h) analyzing plasmid DNA from said high salt medium cultures from step g) for the presence of said recombinant form of a high copy number endogenous plasmid and for the absence of said wild-type high copy number endogenous plasmid,
• i) isolating recombinant Synechococcus sp. strain PCC 7002 from step h) that contain copies of said recombinant form of a high copy number endogenous plasmid and do not contain said wild-type high copy number endogenous plasmid, and
• j) culturing said isolated recombinant Synechococcus sp. strain PCC 7002 from step i) in a high salt medium that does not contain said selective agent for a period of time.