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.

=== Methods ===

* 50 mL linear phase ''Synechocystis'' at OD<sub>730</sub> 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 H<sub>2</sub>O) 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 Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub>, & 1.5% Agar

=== Results ===

* <br>

=== Discussion ===

* We need check that we have all the necessary reagents.
* 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.
<blockquote>''Insanity is doing the same thing over and over again and expecting different results.'' - Albert Einstein</blockquote>

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