Team:Warsaw/Calendar-Main/4 August 2008

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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

  1. 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):
00.10.250.50.751
251.5581.4691.5871.491.5661.311
501.4251.4351.5241.0550.9200.935
751.090.9891.4470.9710.9510.992
1000.090.6851.3781.0780.9770.992

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

  1. Spinning
  2. Suspending
  3. Adding of lysis buffer
  4. Boiling
  5. Putting into poliacrylamide gel
  6. Transfer onto nitrocellulose
  7. Blocking
  8. Anti-A antibody binding
  9. Washing
  10. Anti-rabbit antibody binding
  11. Developing with BCIP and NBT
[a photo of the gel is top be put here]

Michał L., Ewa, Marcin

  1. Separate transformant colonies (transformation from previous day) inoculated to liquid LB with kanamycin.
  2. 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).