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From 2011.igem.org

Adrien Lhomme-Duchadeuil

First, an estimation of how much antibiotics is necessary for Bacillus subtilis. Appropriate concentration in antibiotics

• Chloramphemicol : 5-10 µg/mL
• Spec : 50-100 µg/mL
• Tetracyclin: 25 µg/mL
• Kanamycin: 5µg/mL
• Evm(?): 1µg/mL + 25 µg/mL lincomycin (Verify spelling)

Results of electroporation: IT WORKED after three trials!

• Positive control worked
• Negative controls didn't show any colonies except when put in the presence of TetR (qty:12,5 µg/mL or 5µg/mL) which was insufficient => I will re-plate the improbable survivors on plates of different tetracyclin concentrations to test resistance.
• Cm resistant strain have survived in low quantities at both 30 and 5 µg/mL of antibiotics
• It seems that even cuvettes that had an electric arc were able to produce a couple colonies, which means that it doesn't anihilate all the bacteria.

• To come: protocol and table of results

Table of resutls of electroporation using Cao et al. method

tube 1: poration control no DNA (12,5 kV: ARC) CTL <pow>+</pow>: carpet CTL - with Cm (5 and 30 µg/mL): none CTL - with TetR (5 and 12,5 µg/mL): carpet	tube 2: poration control no DNA (10kV: OK) CTL +: carpet CTL - with Cm: none CTL - with TetR: none	tube 3: pHM3::TetR (12,5 kV: OK) TetR(5 and 12,5 µg/mL): carpet	tube 4: pHM3::TetR (10 kV: OK) TetR (12,5 µg/mL): carpet	tube5: pHM3::TetR (12,5 kV: ARC) TetR (5 and 12,5 µg/mL): carpet	tube 6: pHM3::TetR (10 kV: OK) TetR (5 µg/mL): carpet
tube 7: S12::Cm (12,5 kV: ARC) Cm 5 µg/mL: 1 colony Cm 30 µg/mL: 1 colony	tube 8: S12::Cm (10 kV: OK) Cm 30 µg/mL: 4 colonies	tube 9: S12::Cm (12,5 kV: OK) Cm 30 µg/mL: 9 colonies Cm 5 µg/mL: 4 colonies	tube 10: S12::Cm (10 kV: OK) Cm 5 µg/mL: 1 colonie	CTL --: no bacteria no growth