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Team:Hawaii/PCC6803 Electroporation of PCC6803
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| - | + | = Electroporation of PCC6803 = | |
* This experiment can be used to introduce autonomously replicating plasmids to PCC6803. | * This experiment can be used to introduce autonomously replicating plasmids to PCC6803. | ||
| - | + | == Methods == | |
# 50 mL linear phase ''Synechocystis'' at OD<sub>730</sub> 0.5 collected by centrifugation | # 50 mL linear phase ''Synechocystis'' at OD<sub>730</sub> 0.5 collected by centrifugation | ||
| Line 19: | Line 19: | ||
** cultivation: ''Synechocystis'' is photoautotrophically grown in BG11, 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 | ** cultivation: ''Synechocystis'' is photoautotrophically grown in BG11, 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 | ||
| - | + | == Results == | |
* <br> | * <br> | ||
| - | + | == 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. | *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. | * 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. | ||
Revision as of 04:02, 24 June 2008
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Contents |
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 photoautotrophically grown in BG11, for BG11 agar plates, BG11 supplemented with 10mM N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (pH 8), 12mM Na2S2O3, & 1.5% Agar
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.
- Ludwig, Alfred, “Transformation and gene replacement in the facultatively chemoheterotrophic, unicellular cyanobacterium Synechocystis sp. PCC6714 by electroporation,” Appl Microbiol Biotechnol (2008) 78:729–735
Insanity is doing the same thing over and over again and expecting different results. - Albert Einstein
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