Team:Warsaw/Calendar-Main/18 June 2008

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Latest revision as of 14:55, 26 October 2008


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Preparation of constructs: OmpA_alpha and OmpA_omega

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.

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

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

Blue/white and rifampicin test #2

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

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-<h3>Preparation of constructs with OmpA protein fusions</h3>
+<h3>Preparation of constructs: OmpA_alpha and OmpA_omega</h3>
 <h4>Michał K.</h4>
 <ol><li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177>pACYC177</a>.</li>
@@ Line 12: / Line 12: @@
 <li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of <i>E.coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> with ligation products.</li>
 <li>Plating transformants on LB+tetracycline.</li></ol>
+<a name="fig1"> <img src="https://static.igem.org/mediawiki/2008/c/c9/Ompa_Alpha_Omega_Cleanup_WAW.jpg" width=300/> </a>
+<var> <b>Fig. 1. </b>OmpA_alpha (lane 2) and OmpA_omega (lane 3) digest products.</var>
+<a name="fig2"><img src="https://static.igem.org/mediawiki/2008/5/5f/PACYC177_digest_WAW.jpg" width=300/></a>
+<var><b>Fig. 2.</b> pACYC177 digested with NdeI and BamHI (lane 2).</var>
-<h3> Blue/white and rifampicin test </h3>
+<h3> Blue/white and rifampicin test #2</h3>
 <h4>Michał L., Ewa, Piotr</h4>
 <table width="100%" class="month">
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-<a name="fig1"> <img src="https://static.igem.org/mediawiki/2008/c/c9/Ompa_Alpha_Omega_Cleanup_WAW.jpg" width=300/> </a>
-<var> <b>Fig. 1. </b>OmpA_alpha (lane 2) and OmpA_omega (lane 3) digest products.</var>
-<a name="fig2"><img src="https://static.igem.org/mediawiki/2008/5/5f/PACYC177_digest_WAW.jpg" width=300/></a>
-<var><b>Fig. 2.</b> pACYC177 digested with NdeI and BamHI (lane 2).</var>
 </td></tr></table>
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