Team:Hawaii/Initial PCC6803 Transformation

= Transformation of Synechocystis sp. PCC 6803 =

Protocol by Arizona State University

 * 1) Grow Synechocystis on BG-11 + glucose medium to OD730 = 0.5.
 * 2) Transfer 1.5 ml of culture to a sterile microcentrifuge tube.
 * 3) Spin cells at 7000 rpm for 2 minutes in a balanced microcentrifuge.
 * 4) Remove supernatant by pipetting.
 * 5) Add 200 µl of fresh BG-11 medium to the cells and resuspend cells.
 * 6) Add 2 µl of the [our] plasmid and shake the tube several times.
 * 7) Incubate at room temperature for 30 minutes.
 * 8) Place a sterile filter on top of an agar plate that contains BG-11 + 5 mM glucose (50 ml per plate because it will take a long time for colonies to appear). Plate the cell suspension on the filter, spread it, and let it dry. The plate will be incubated in the light at 30 °C. The filter will be transferred the next day to a BG-11/glucose plate that contains [antibiotic].

Questions

 * 1) Concentration of plasmid?
 * 2) Temperature for growth in step 1? Light conditions?
 * 3) Plate how much cells?
 * 4) Use how much antibiotic?
 * Some possible answers from Mermet-Bouvier-CM-1993
 * Antibiotics added under the agar plates (I don't know why they add under the plate, perhaps some of the antibiotics are too heat sensitive) to a final concentration of: 10ug/mL Chloramphenicol, 50ug/mL Kanamycin, or 5ug/mL streptomycin.

Reference
“Experiment II: Genetic Manipulation of Synechocystis sp. PCC 6803.” Genetic Engineering and Society. 16 Aug 2007. Arizona State University. 01 June 2008. Available.

Protocol by Shao, et al

 * 1) Synechocystis sp. strain PCC 6803 cells was grown routinely in BG-11 liquid and solid media at 30oundefinedC with constant illumination (25 μmol photons m−2 s−1 from cool-white fluorescent lamps) to OD730 = ~0.6. Cells were harvested by centrifugation at 4,500 x g for 10 min at room temperature. The cell pellet was resuspended in fresh BG-11 medium at a density of 109 cells/ml and transformated immediately.
 * 2) Plasmid DNA (1 μg plasmid in 10 μl 10 mM Tris-HCl [pH 8.5]) was added to 1 ml of cells, gently mixed, and incubated for 1 hour without agitation at 30oC with illumination.
 * 3) Cells were spread on 50-ml BG-11 agar plates (0.1 ml per plate) and incubated under illumination for 2 days at 30°C. At this stage, the appropriate selective agent (kanamycin) was added by lifting the agar slab with an ethanol-flamed spatula and dispensing 50 μl of a solution containing kanamycin (10 mg ml−1) underneath. After 14 days, transformant colonies were isolated and purified by restreaking three times in the presence of antibiotics as above.

Reference
Shao CY, Howe CJ, Porter AJR, and Glover LA. “Novel Cyanobacteria Biosensor for Detection of Herbicides.” Applied Environmental Microbiology. 2002. 68(10): 5026-5033. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=126403

Protocol used

 * 1) Grew PCC6803 to OD730 = 0.2 and 1.5 (ideal is OD730 = 0.5-0.8)
 * 2) Transferred 0.75ml cells to sterile eppendorf tube
 * 3) Centrifuged 8 min @ 7000g for 8 minutes at room temperature. OD=1.5 sample was centrifuged for an additional 2 min @ 1000g.
 * 4) Cells were resuspended in 100 &mu;l BG-11
 * 5) Incubated at 30C w/ illumination (36 &mu;l photoms m2 s2 for 1 hour
 * 6) Added 1 mL BG-11
 * 7) Incubated at 30C w/ illumination for 2 days
 * 8) Plated 0.1 ml cells on  BG-11 + spectinomycin + streptomycin plate


 * Original cell density determined by plating 0.2 ml 1/1, 1/10, 1/100 dilutions of cells on BG-11 plates.
 * PCC6803 is not naturally resistant to sp + st.

Results
FAILED. No transformants were detected after incubating in the presence of CO2 at 37C for 18 days. Dots observed are moisture droplets reflecting light.



Discussion
Althought Synechocystis sp. PCC6803 is naturally competent, plasmids that cannot homogolously recombine are not retained. Transformation is not an effectively way to introduce exogenous DNA into PCC6803.