Team:Warsaw/Calendar-Main/6 August 2008

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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):
ampicillin concentration (μg/mL):	0	0.1	0.25	0.5	0.75	1
25	1.054	1.154	1.051	0.99	1.096	0.896
50	0.94	0.891	1.123	0.847	0.924	0,81
75	0.5	0.63	0.743	0.782	0.631	0.64
100	0.02	0.396	0.563	0.678	0.602	0.611

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 ompt 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.

Preparing pACYC177+OmpA_omega_deltaA construct

Michał K.

Inoculation of cultures from colonies grown on plate with pACYC177+OmpA_omega_deltaA transformation.