Abstract
Synechococcus sp. PCC 7002 is an ideal model cyanobacterium for functional genomics and biotechnological applications through metabolic engineering. A gene expression system that takes advantage of its multiple, endogenous plasmids has been constructed in this cyanobacterium. The method involves the integration of foreign DNA cassettes with selectable markers into neutral sites that can be located on any of the several endogenous plasmids of this organism. We have exploited the natural transformability and powerful homologous recombination capacity of this organism by using linear DNA fragments for transformation. This approach overcomes barriers that have made the introduction and expression of foreign genes problematic in the past. Foremost among these is the natural restriction endonuclease barrier that can cleave transforming circular plasmid DNAs before they can be replicated in the cell. We describe herein the general methodology for expressing foreign and homologous genes in Synechococcus sp. PCC 7002, a comparison of several commonly used promoters, and provide examples of how this approach has successfully been used in complementation analyses and overproduction of proteins with affinity tags.
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Acknowledgments
This research has been partly supported by grant MCB-0519743 from the National Science Foundation as well as by subcontract 00001041 from Princeton University to D.A.B. The primary award for this subcontract is Multi-University Research Initiative (MURI) grant FA-9550-05-1-0365 from the U. S. Air Force Office of Scientific Research to Dr. G. Charles Dismukes, Department of Chemistry, Princeton University.
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Xu, Y., Alvey, R.M., Byrne, P.O., Graham, J.E., Shen, G., Bryant, D.A. (2011). Expression of Genes in Cyanobacteria: Adaptation of Endogenous Plasmids as Platforms for High-Level Gene Expression in Synechococcus sp. PCC 7002. In: Carpentier, R. (eds) Photosynthesis Research Protocols. Methods in Molecular Biology, vol 684. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-925-3_21
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