Team:IIT Madras/Project



=Project Details= StressKit: A BioBrick library of Lac-repressed sigma-24, sigma-28, sigma-32 and sigma-38 promoters for Escherichia coli.

Protocols
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Procedure for growth curve (Before standardisation)
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 * Inoculate the LB broth with a single colony and grow it as an overnight culture.
 * Add 50µg/ml spectinomycin for the flask containing the k12z1 strain and 100µg/ml of ampicillin for all the other flasks containing the constructs
 * We transfer X% inoculum such that the cells reach the OD of 0.1 in the M9 minimal media, transfer 1% of the overnight LB culture into the flasks containing the plasmids grown in M9 minimal media.
 * Take two sets of are samples (1ml each) from each flask for OD and CFP measurements.
 * Samples were taken hour.
 * To measure OD we centrifuge the sample for 10 min at 10000 rpm
 * Then resuspend them in saline and take the reading using a UV spectrometer @600nm
 * Saline was used as blank solution
 * For YFP measurements 1 ml of the sample was taken and measurement was done at a 434nm excitation and 470nm emission in fluorescence microscope

Growth curve (standardized)

 * Inoculate the LB broth with a single colony and grow it for 6hours.
 * Add 100µg/ml spectinomycin for the flask containing the k12z1 strain and 50µg/ml of ampicillin for all the other flasks
 * Transfer 1% of the overnight LB culture into the flasks containing the plasmids grown on M9
 * Take two sets of are samples (1ml each) from each flask for OD and YFP measurements.
 * Samples should be taken hour( up to 8 hrs )
 * The OD was measured without centrifugation @600nm
 * M9 medium was used as blank solution
 * The YFP was measured at 514nm excitation and 527nm mission in fluorescence microscope.

Standardized protocol for stress

 * Inoculate the LB broth with a single colony and grow it for 6hours.
 * Two flasks were assigned for each plasmid. One flask with IPTG (0.25mm) and the other without IPTG.
 * Transfer 1% of the overnight LB culture into the flasks containing the plasmids grown on M9
 * Allow it to grow for 4hrs
 * Then we stress it using the following conditions:
 * 500mM NaCl in Minimal Media M9
 * 100microM H2O2 in Minimal Media M9
 * Heat Shock by keeping in 42 degrees for 120 seconds
 * PH of 5.5 achieved by adding 4.150 ml of 3% HCl in 50 ml Minimal M9
 * Starvation stress: Pellet out cells in early exponential phase and re suspend them in M9 media without glucose. This is called severe CARBON - LESS stress
 * The first sample was taken after 30min of stressing.
 * Samples were taken every subsequent hour (up to 5hours)
 * The OD was measured at 600nm using UV spectrometer.
 * The YFP was measured at 514nm excitation and 527nm emission in fluorescence microscope.

Preparation of M9

 * Prepare 10x m9 stock solution

For 50 ml of M9 media

 * 10x m9        -5 ml
 * 20% glucose   - 1ml
 * MgSO4         - 0.1 ml


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