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).
Checking if OmpA_omega_A_alpha gives ampicillin resistance
Piotr
Inoculation OmpA_omega_A_alpha from various IPTG concentrations (in mmol/mL) (0, 0.1, 0.25, 0.5, 0.75, 1) into same IPTG concentrations, but with various ampicillin concentrations (in mmol/mL)(25, 50, 75, 100) in ratio: 1:50.
Measurement of bacterial culture growth (OD) in the evening:
| ampicillin concentration (μg/mL): | IPTG concentration (mmol/mL): |
|---|
| 0 | 0.1 | 0.25 | 0.5 | 0.75 | 1 |
|---|
| 25 | 1.558 | 1.469 | 1.587 | 1.49 | 1.566 | 1.311 |
|---|
| 50 | 1.425 | 1.435 | 1.524 | 1.055 | 0.920 | 0.935 |
|---|
| 75 | 1.09 | 0.989 | 1.447 | 0.971 | 0.951 | 0.992 |
|---|
| 100 | 0.09 | 0.685 | 1.378 | 1.078 | 0.977 | 0.992 |
|---|
Inoculation of OmpA_omega_A_alpha into various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75 mmol/mL (replication is necessary)
Checking OmpA_omega_A_alpha and OmpA_A_alpha expression
Piotr
- Spinning
- 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
[a photo of the gel is top be put here]
Michał L., Ewa, Marcin
- Separate transformant colonies (transformation from previous day) inoculated to liquid LB with kanamycin.
- Inoculation of pACYC177+OmpA_alpha and pACYC177+OmpA_omega - liquid LB with kanamycin.
Michał K.
Separate transformant colonies (tranformations from previous day) inoculated to liquid LB with kanamycin
Checking for presence of A on the cell membrane
Piotr
Inoculation of omp_A_alfa, omp_Z_alfa and omp_omega_A_alfa (with and without induction).
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