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 trunced 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.

Piotr:

Checking if degradation of fusion with OmpA is a result of Top10 proteases' activity (lon, iompt)

Inoculation of OmpA_omega_A_alpha and OmpA_A_alpha with and without inductor (0,5IPTG) in Rosetta.