Team:Warsaw/Calendar-Main/18 June 2008


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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.

Fig. 1. OmpA_alpha (lane 2) and OmpA_omega (lane 3) digest products.

Fig. 2. pACYC177 digested with NdeI and BamHI (lane 2).

Counting of blue colonies:

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