Team:Warsaw/Calendar-Main/5 August 2008

Cloning of truncated fragment of protein A (&Delta;A) 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_&Delta;A_alpha gives ampicillin resistance Piotr, Weronika

Inoculation OmpA_omega_&Delta;A_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.

<h4 style="text-align: center">Measurement of bacterial culture growth (OD) in the evening:

Inoculation of <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>OmpA_omega_&Delta;A_alpha</a> into various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75 mmol/mL (repetition is necessary)

Checking <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>OmpA_omega_&Delta;A_alpha</a> expression Piotr, Weronika

 Spinning.</li> Suspending.</li> Adding of lysis buffer.</li> Boiling.</li> Putting into poliacrylamide gel.</li> Transfer onto nitrocellulose.</li> Blocking.</li> Anti-A antibody binding.</li> Washing.</li> Anti-rabbit antibody binding.</li> Developing with BCIP and NBT (<a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/5_August_2008#fig1">Fig. 1.</a>). </li> </ol>

<img src="http://2008.igem.org/wiki/images/e/ea/Western1_WAW.jpg" width=400/></a> 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 <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-deltaA>pACYC177+OmpA_omega_&Delta;A</a> construct Michał K. <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#electrotransform">Transformation</a> of E. coli <a href="http://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> strain with ligation. </li> Transformants plating on LB + kanamycin. </li></ol>