Team:Warsaw/Calendar-Main/30 July 2008

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  1. Isolation of plasmids from cultures inocluated on previous day.
  2. Control digest of isolated plasmids (pACYC177+omega_deltaA) with HindIII and SacI (7 from 8 colonies confirmed).

Checking if OmpA_omega_A_alpha gives ampicillin resistance
Piotr

Inoculation of OmpA_omega_A_alpha from various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75, 1 mM into same IPTG concentrations, but with various ampicillin concentrations (25, 50, 75, 100 mM) in ratio 1:50.

Bacterial growth (OD) measurement in the evening:

ampicillin concentration (μg/mL):IPTG concentration (mmol/mL):
00.10.250.50.751
251.0541.1541.0510.991.0960.896
500.940.8911.1230.8470.9240,81
750.50.630.7430.7820.6310.64
1000.020.3960.5630.6780.6020.611

Conclusion: we obtained the best induction using 0.5 - 0.25 mM IPTG. Best ampicillin concentration is 50 - 75 μg/ml.

Checking if degradation of fusion with OmpA is a result of the activity of lon and iompt proteases. (present in top10)
Piotr

Test was conducted in E.coli Rosetta strain expressing omp_omega_A_alfa (with and without induction) and omp_A_alfa.

Transformation of Rosettas with omega_A_alpha and A_alpha.

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