Latest revision as of 18:33, 30 June 2008

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Electroporation of PCC6803

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

Methods

50 mL linear phase Synechocystis at OD₇₃₀ 0.5 collected by centrifugation
wash cells three times with 1mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer pH 7.5
remove supernatant, resuspend in 100 uL of the remaining liquid by vortexing
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)
IMMEDIATELY after electric pulse, cells resuspended in 1 mL BG11 & mixed with 50 mL BG11 in an Erlenmeyer flask
cells incubated 5 days under **specified growth conditions
50 mL culture collected by centrifugation, resuspended in 500uL of remaining liquid, mixed with 5mL BG11 soft agar (BG11 + 0.75% agar)
Pour on plates with 25 mL BG11 agar with antibiotic
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)
- A PCC6803 cell density test was performed and aligned with a growth curve (Richardson 1983).

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

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

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

[http://manoa.hawaii.edu/ ][http://manoa.hawaii.edu/ovcrge/ ][http://www.ctahr.hawaii.edu ]

@@ Line 17: / Line 17: @@
 # 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:
+<strong> Cultivation </strong>
-**''Synechocystis'' is photoautotrophically grown in BG11 at 30C under white light (Chauvat 1986)
+*''Synechocystis'' is photo-autotrophically grown in BG11 at 30&deg;C under white light (Chauvat 1986)
-**for BG11 agar plates, BG11 supplemented with 10mM N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (pH 8), 12mM   Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub>, & 1.5% Agar
+*Cells grown to 10^9 cells/mL (Mermet-Bouvier)
+**A [[Team:Hawaii/PCC6803 cell density| PCC6803 cell density]]  test was performed and aligned with a growth curve (Richardson 1983).
+[[Image:Growth_curve.JPG|right|thumbnail|300 px|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).]]
+<strong>Plasmid Concentration</strong>
+*Use estimates from gel found in [[Team:Hawaii/Large-Scale Preparation of Plasmid from E. coli| Large-Scale Preparation of Plasmid from E. coli]]
+*Use calculation from UV spectrometric reading found in [[Team:Hawaii/Large-Scale Preparation of Plasmid from E. coli| Large-Scale Preparation of Plasmid from E. coli]]
 == Results ==
@@ Line 31: / Line 36: @@
 *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.
 <blockquote>''Insanity is doing the same thing over and over again and expecting different results.'' - Albert Einstein</blockquote>
 {{Team:Hawaii/Footer}}

Team:Hawaii/PCC6803 Electroporation of PCC6803

From 2008.igem.org