Team:Warsaw/Calendar-Main/5 August 2008

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Cloning of truncated fragment of protein A (ΔA)

Emilia

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

Checking if OmpA_omega_ΔA_alpha gives ampicillin resistance

Piotr, Weronika

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):
ampicillin concentration (μg/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_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 expression

Piotr, Weronika

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 (Fig. 1.).

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

Preparing pACYC177+OmpA_omega_ΔA construct

Michał K.

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