Abstract
Synechococcus elongatus PCC 7942 is an excellent model system for studying the molecular mechanism of the circadian clock in cyanobacteria. The “plastic” genetic characteristics of this organism have facilitated the development of various methods for mutagenesis of its chromosome. These methods are based on homologous recombination between the chromosome and foreign DNA, introduced to the cyanobacteria by either transformation or conjugation. Here we describe different approaches to mutagenize the chromosome of S. elongatus, including insertional mutagenesis, hit-and-run allele replacement, rps12-mediated gene replacement, and regulated expression of genes from ectopic sites, the neutral sites of the S. elongatus genome.
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References
Golden, S. S., Brusslan, J., and Haselkorn, R. (1987) Genetic engineering of the cyanobacterial chromosome. Methods Enzymol. 153, 215–231.
Golden, S. S., and Sherman, L. A. (1984) Optimal conditions for genetic transformation of the cyanobacterium Anacystis nidulans R2. J. Bacteriol. 158, 36–42.
Elhai, J., and Wolk, C. P. (1988) Conjugal transfer of DNA to cyanobacteria. Methods Enzymol. 167, 747–754.
Andersson, C. R., Tsinoremas, N. F., Shelton, J., et al. (2000) Application of bioluminescence to the study of circadian rhythms in cyanobacteria. Methods Enzymol. 305, 527–542.
Matsuoka, M., Takahama, K., and Ogawa, T. (2001) Gene replacement in cyanobacteria mediated by a dominant streptomycin-sensitive rps12 gene that allows selection of mutants free from drug resistance markers. Microbiology 147, 2077–2087.
Takahama, K., Matsuoka, M., Nagahama, K., and Ogawa, T. (2004) High-frequency gene replacement in cyanobacteria using a heterologous rps12 gene. Plant Cell Physiol. 45, 333–339.
Bustos, S. A., and Golden, S. S. (1991) Expression of the psbDII gene in Synechococcus sp. strain PCC 7942 requires sequences downstream of the transcription start site. J. Bacteriol. 173, 7525–7533.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Golden, S. S., and Haselkorn, R. (1985) Mutation to herbicide resistance maps within the psbA gene of Anacystis nidulans R2. Science 229, 1104–1107.
Mori, T., Binder, B., and Johnson, C. H. (1996) Circadian gating of cell division in cyanobacteria growing with average doubling times of less than 24 hours. Proc. Natl. Acad. Sci. USA 93, 10,183–10,188.
Tsinoremas, N. F., Kutach, A. K., Strayer, C. A., and Golden, S. S. (1994) Efficient gene transfer in Synechococcus sp. strains PCC 7942 and PCC 6301 by interspecies conjugation and chromosomal recombination. J. Bacteriol. 176, 6764–6768.
Kondo, T., Tsinoremas, N. F., Golden, S. S., Johnson, C. H., Kutsuna, S., and Ishiura, M. (1994) Circadian clock mutants of cyanobacteria. Science 266, 1233–1236.
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Clerico, E.M., Ditty, J.L., Golden, S.S. (2007). Specialized Techniques for Site-Directed Mutagenesis in Cyanobacteria. In: Rosato, E. (eds) Circadian Rhythms. Methods in Molecular Biology™, vol 362. Humana Press. https://doi.org/10.1007/978-1-59745-257-1_11
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DOI: https://doi.org/10.1007/978-1-59745-257-1_11
Publisher Name: Humana Press
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Online ISBN: 978-1-59745-257-1
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