Team:Warsaw/Calendar-Main/5 August 2008

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Cloning of truncated fragment of protein A (ΔA)

Emilia

  1. Isolation of plasmids from cultures inoculated on previous day.
  2. Control digest with SacI and BamHI (BamHI buffer).
  3. Gel electrophoresis - proper clones found for both products of ligation.

Checking if OmpA_omega_ΔA_alpha gives ampicillin resistance

Piotr

Inoculation OmpA_omega_ΔA_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_ΔA_alpha into various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75 mmol/mL (repetition is necessary)

Checking OmpA_omega_ΔA_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 (Fig. 1.).
Fig. 1.Protein A detection in bacterial lysates.
1 - protein marker,
2 and 3 - E coli without plasmid,
4 - Omp_omega_A_alpha + 0,5 mM IPTG,
5 - E coli without plasmid,
6 - Omp_omega_A_alpha + 0,5 mM IPTG,
7 - Omp_omega_A_alpha without inducer.


Preparing pACYC177+OmpA_omega_ΔA construct

Michał K.

  1. Transformation of E. coli TOP10 strain with ligation.
  2. Transformants plating on LB + kanamycin.