1. Physical specification: The promoters must conform to the BioBrick format, and expression must be quantifiable using an existing BioBrick fluorescent reporter. This ensures that our library can be used immediately in conjunction with Registry parts.
2. Functional specification: The promoters must be 'gated' by the LacI repressor, so they remain switched off unless a Lac inducer such as IPTG is present. In the presence of IPTG, the promoter must behave like a 'bare' sigma-factor-dependent promoter, expressing only under specific stress conditions.
3. Modularity: The entire library of promoters must have the same design format; each promoter must involve a minimal DNA region, and must not require any transcription factors apart from LacI and the relevant sigma factor; and the promoters must have minimal cross-talk. This ensures that that different sigma-dependent responses can be directly compared, and if necessary combined into the same device, with no additional components required.

Procedure for growth curve (Before standardisation)

• 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

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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