Team:Hawaii/PCC6803 Electroporation of PCC6803

= 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&deg;C under white light (Chauvat 1986)
 * Cells grown to 10^9 cells/mL (Mermet-Bouvier)
 * A PCC6803 cell density  test was performed and aligned with a growth curve (Richardson 1983).

Plasmid Concentration
 * Use estimates from gel found in Large-Scale Preparation of Plasmid from E. coli
 * Use calculation from UV spectrometric reading found in Large-Scale Preparation of Plasmid from E. coli

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.