Checking the expression of omp_omega_A_alpha and omp_A_alpha
Piotr
Inoculation of omp_omega_A_alpha and omp_A_alpha with inductor (0,5 mmol/mL IPTG) and negative control without IPTG.
Checking if omp_omega_A_alpha gives ampicillin resistance
Piotr
- Inoculation of omp_omega_A_alpha into various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75, 1 mmol/mL
1. Optimization of PCR to obtain truncated fragment of protein A DNA
Primers:
AL+SacI
AP+NotI
Elongation time: 30s
- Optimization of annealing temperature (gradient from 55°C to 75°C)
- Optimization of number of cycles(15, 20, 25, 30, 35)
2. PCR to obtain truncated A protein DNA fragment
Primers:
AL+SacI
AP+NotI
Elongation time: 30s
Annealing temperature: 60°C
20 cycles
3. Gel electrophoresis and isolation of 250 bp band.
4. Digest of isolated PCR product, pACYC177+OmpA_alpha and pACYC177+OmpA_omega with NotI and SacI.
5. Clean-up of digestion reaction.
6. Gel electrophoresis for estimation of DNA concentration.
7. Overnight ligation: pACYC177+OmpA_alpha + deltaA and pACYC177+OmpA_omega + deltaA.
Checking if degradation of fusion with OmpA is a result of Top10 proteases' activity (lon, iompt)
Piotr:
Inoculation of OmpA_omega_A_alpha and OmpA_A_alpha with and without inductor (0,5 mm/mL IPTG) in E. coli Rosetta strain.
1. Isolation of plasmids from cultures inocluated on previous day.
2. Control digest of isolated plasmids with BamHI and SacI (we confirmed 4 colonies but A in our constructs turned out to be trunctated and contain only one from two highly similar domains).
"
