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): |
|---|
| 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 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.
Michał K.
- Transformation of E. coli TOP10 strain with ligation products (pACYC177+OmpA_alpha + deltaA and pACYC177+OmpA_omega + deltaA).
- Transformants plating on LB + kanamycin.
Checking whether degradation of the fusions with OmpA is caused by 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.
- Spinnign
- Suspending
- Adding of lysis buffer
- Boiling
- Putting into poliacrylamide gel
- Transfer onto nitrocellulose
- Blocking
- Anti-A antibody binding
- Washing
- Anti-rabbit antibody binding
- Developing with BCIP and NBT
[photo of the gel is to be placed here]
We didn't observe differences in espression and degradation in Rosettas nor in TOP10. Therefore we suppose that degradation of the fusions is caused by other factor than Lon and OmpT proteases.
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