Team:Warsaw/Calendar-Main/18 June 2008

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Preparation of constructs with OmpA protein fusions

Michał K.

Isolation of pACYC177.
Digest of purified pACYC177 with NdeI and BamHI (Tango 2x buffer).
Gel electrophoresis (Fig. 2) and isolation of proper band (3200 bp).
Clean-up of OmpA_alpha and OmpA_omega digest products (Fig. 1).
Ligation of purified DNA (pACYC177 with OmpA_alpha and OmpA_omega DNA fragments).
Transformation of E.coli TOP10 with ligation products.
Plating transformants on LB+tetracycline.

Blue/white and rifampicin test

Michał L., Ewa, Piotr

Counting of blue colonies:

Strain	% of blue colonies
pMPMT5	96%
pMPMT5-AID	98%
pMPMT5-AIDT7	61%
pMPMT5-AID+T7	1%

Results of rifampicin test

Strain	Inducer / repressor	Number of colonies on rifampicin	OD
Top10	none	3	2.91
Top10 pMPM-AID	0.1% Arabinose	72	2.7
Top10 pMPM-AID	none	8	2.58
Top10 pMPM-AID+T7	0.1% Arabinose	148	1.88
Top10 pMPM-AID+T7	none	6	2.52
Top10 pMPM-AIDT7	0.1% Arabinose	17	2.74
Top10 pMPM-AIDT7	none	21	2.59

AID in transcription fusion works like AID, but induce slower growth than AID;
AID in translation fusion do not induce rifampicin resistance mutation Fig. 1. OmpA_alpha (lane 2) and OmpA_omega (lane 3) digest products. Fig. 2. pACYC177 digested with NdeI and BamHI (lane 2).