Team:Hawaii/PCC6803 Electroporation of PCC6803

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Contents

Electroporation of PCC6803

  • This experiment can be used to introduce autonomously replicating plasmids to PCC6803.

Methods

  1. 50 mL linear phase Synechocystis at OD730 0.5 collected by centrifugation
  2. wash cells three times with 1mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer pH 7.5
  3. remove supernatant, resuspend in 100 uL of the remaining liquid by vortexing
  4. 60 uL of cell suspension mixed with 0.1-16 ug DNA (dissolved in H2O) and electroporated with Biorad gene pulser (25uF capacitor used, time constant varied buy changing resistors (100, 200, 400, & 600 OHMS for time constant 2.5, 4.8, 9, 13 ms, respectively, electric field varied from 0-12kV cm-1)
  5. IMMEDIATELY after electric pulse, cells resuspended in 1 mL BG11 & mixed with 50 mL BG11 in an Erlenmeyer flask
  6. cells incubated 5 days under **specified growth conditions
  7. 50 mL culture collected by centrifugation, resuspended in 500uL of remaining liquid, mixed with 5mL BG11 soft agar (BG11 + 0.75% agar)
  8. Pour on plates with 25 mL BG11 agar with antibiotic
  9. determine number of viable cells after electroporation: 10^-4 dilution made before collecting the culture, 50 uL of this dilution plated with out selection, incubate 14 days, count colonies. Total viable cells (colonies grown on non-selective plates * 10^7) and total number transformants (colonies on selective plates) determined

Cultivation

  • Synechocystis is photo-autotrophically grown in BG11 at 30°C under white light (Chauvat 1986)
  • Cells grown to 10^9 cells/mL (Mermet-Bouvier)
Growth curve (semi-log plot) of Synechocystis sp. PCC6803 cells inoculated in BG-11 medium (open circles), marine medium (closed circles), and twice normal marine medium (closed boxes).

Plasmid Concentration

Results


Discussion

  • 40uL of cells at 10^9 cells/mL were mixed with 5 uL of plasmid DNA solution (1ug/uL). Electroporation was performed at field strength of 12kV/cm at a constant of 5ms, with no cell death detected. (Mermet-Bouvier)*used for PCC6803 and other strains.
  • This protocol is adapted from a protocol for PCC6714, which is not naturally competent. However it seems we can use this for PCC6803 so we can obtain autonomously replicating plasmids.

References

  • Ludwig, Alfred, “Transformation and gene replacement in the facultatively chemoheterotrophic, unicellular cyanobacterium Synechocystis sp. PCC6714 by electroporation,” Appl Microbiol Biotechnol (2008) 78:729–735
  • Chauvat, F, "A host-vector system for gene cloning in the cyanobacterium Synechocystis PCC6803," Mol Gen Genet (1986) 204:185-191.
  • Richardson, D.L., "Synechocystis PCC6803: a euryhaline cyanobacterium," FEMS Microbiology Letters 18 (1983) 99-102.
Insanity is doing the same thing over and over again and expecting different results. - Albert Einstein


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