Team:Hawaii/Initial PCC6803 Transformation

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# Plate how much cells?<br>
# Plate how much cells?<br>
# Use how much antibiotic?<br>
# Use how much antibiotic?<br>
 +
* 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 ====
==== Reference ====

Revision as of 04:59, 6 June 2008

Contents

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 [http://photoscience.la.asu.edu/photosyn/courses/BIO_343/lab/Experiment-II.html].


Protocol by Shao, et al

  1. Synechocystis sp. strain PCC 6803 cells was grown routinely in BG-11 liquid and solid media at 30oC 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


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