Team:EPF-Lausanne/Our Project/T7 promoter variants/lysis
From 2011.igem.org
Lysis Selection System
Lysis selection system Main | Lysis Characterization | DNA Recovery | DNA Selection | T7 Promoter Variants
Introduction
We cloned the Berkeley Lysis cassette (BBa_K112808) under control of the T7 promoter into a low copy number plasmid (BBa_I739202, known as [http://partsregistry.org/Part:pSB3K1 pSB3K1])
We tested lysis of cells haboring this plasmid in a platereader experiment. Lysis resulted in a drop in optical density after induction with IPTG. We observed a strong dependence on IPTG concentration.
Experimental Setup
1) We made liquid cultures of BL21 cells containing the plasmids with the lysis cassette driven by a T7 promoter. These plasmids have Kanamycin resistance, so the cultures were made using 5 mL of LB medium with 5 uL of 100 ug/uL Kanamycin and grown overnight.
2) We made 1:10 dilutions of cells to LB medium by adding 100 uL of cells to 900 uL of LB with 1 uL of Kanamycin. From this 1 mL mix, we pipetted 100 uL into the wells of a 96 well plate.
3) We covered each well with 20 uL of mineral oil to avoid evaporation. Then we set the platereader to shake continuously and take optical density measurements at 600 nm wavelength every ten minutes.
4) We waited an hour and a half (approximately) until the cells had reached their log-linear growth phase. Then we took out the plate and added 1 uL of IPTG to each well. We made triplicate data for each concentration of IPTG (0 uL for negative control, 50 uL, 100 uL, 250 uL, and 500 uL).
5) We set the plate again into the platereader, ran it for 12 hours with continous shaking. Optical density measurements were made every ten minutes.
Results