An Improved Natural Transformation Protocol for the Cyanobacterium Synechocystis sp. PCC 6803
Open accessAbstract
The naturally transformable cyanobacterium Synechocystis sp. PCC 6803 is a widely used chassis strain for the photosynthetic production of chemicals. However, Synechocystis possesses multiple genome copies per cell which means that segregating mutations across all genome copies can be time-consuming. Here we use flow cytometry in combination with DNA staining to investigate the effect of phosphate deprivation on the genome copy number of the glucose-tolerant GT-P sub-strain of Synechocystis 6803. Like the PCC 6803 wild type strain, the ploidy of GT-P cells grown in BG-11 medium is growth phase dependent with an average genome copy number of 6.05 ± 0.27 in early growth (OD740 = 0.1) decreasing to 2.49 ± 0.11 in late stationary phase (OD740 = 7). We show that a 10-fold reduction in the initial phosphate concentration of the BG-11 growth medium reduces the average genome copy number of GT-P cells from 4.51 ± 0.20 to 2.94 ± 0.13 and increases the proportion of monoploid cells from 0% to 6% after 7 days of growth. In addition, we also show that the DnaA protein, which unusually for bacteria is not required for DNA replication in Synechocystis, plays a role in restoring polyploidy upon subsequent phosphate supplementation. Based on these observations, we have developed an alternative natural transformation protocol involving phosphate depletion that decreases the time required to obtain fully segregated mutants.
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An even simpler transformation approach has been proposed that does not require any specific inhibitor(s); it relies on spirulina's innate transformation capacity, double homologous recombination, and further antibiotic selection.
“An even simpler transformation approach has been proposed that does not require any specific inhibitor(s). This methodology relies on spirulina’s innate transformation capacity, double homologous recombination, and further antibiotic selection [116,117].”
From Spirulina/Arthrospira/Limnospira—Three Names of the Single Organism (2024) · cites this paper as [116]
The Introduction describes natural transformation and double homologous recombination in Synechocystis sp. PCC 6803, and the Results describe a phosphate-deprivation protocol followed by kanamycin selection (page numbers unavailable). The cited paper does not establish this approach for Spirulina; its tested organism is Synechocystis. The described protocol uses no specific inhibitor, but phosphate deprivation is an important condition omitted from the claim. (LLM, conf=0.88)
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- Authors
- Pope, M. A., Hodge, J. A., & Nixon, P. J.
- Affiliations
- Imperial College London (GB)
- Corresponding author
- Nixon, Peter J.
- ORCID
- Nixon, Peter J.
- Venue
- Frontiers in Plant Science
- Publisher
- Frontiers Media SA
- Published
- 2020-04-15
- Volume
- 11
- Pages
- 372-372
- ISSN
- 1664-462X (ISSN-L)
- PMC
- PMC7174562
- OpenAlex
- W3016577172
- Cited by
- 42
- References
- 44
- Citations by year
- 2026: 8 · 2025: 13 · 2024: 4 · 2023: 7 · 2022: 4 · 2021: 5 · 2020: 1
- FWCI
- 2.49
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Frontiers in Plant Science (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Spiral (Imperial College London) (submittedVersion, cc-by)
- Funders
- Biotechnology and Biological Sciences Research Council
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Photosynthetic Processes and Mechanisms
- Keywords
- Synechocystis sp. PCC 6803, natural transformation, genome copy number, polyploidy, phosphate limitation, DnaA protein, DNA replication, flow cytometry
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