Team:Warsaw/Calendar-Main/31 July 2008

Cloning of truncated fragment of protein A (&Delta;A) Michał K.   Transformation of E. coli TOP10 strain with ligation products (pACYC177+OmpA_alpha + &Delta;A and pACYC177+OmpA_omega + &Delta;A).   Transformants plating on LB + kanamycin. 

Cloning of omega_&Delta;A DNA fragment to pACYC177+OmpA_alpha Michał K.

 <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation of plasmids</a> from cultures inocluated on <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/31_July_2008">previous day</a>. </li>  Control <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">digest</a> of isolated plasmids with BamHI and SacI (BamHI buffer). </li> Gel electrophoresis - proper clones founded (<a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/31_July_2008#fig1">Fig. 1.</a>). </li></ol> <img src="http://2008.igem.org/wiki/images/2/2e/31_july.jpg" width=250/></a> Fig. 1. Control SacI/BamHI digests of isolated plasmids 1-4. digested plasmids pACYC177 + OmpA-omega-&Delta;A-alpha 5. Marker

Purification of proteins: Z-alpha and Z-omega Piotr, Emilia Samples were resuspended in PBS, sonicated and centrifuged.</li> Lysis buffer was added separately to pellet and supernatant, then samples were boiled for 10 min. </li> Lysates loaded on 12% poliacrylamide gel (amount relating to 100 μl of OD=1.0 culture).</li> Gel stained with Coomassie Blue. Optimal induction conditions chosen (<a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/31_July_2008#fig2">Fig. 2.</a> and <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/31_July_2008#fig3">Fig. 3.</a>).</li></ol> <img src="http://2008.igem.org/wiki/images/e/e7/July_31_st.jpg" width=350 /></a> Fig. 2. Pellets from Z-omega induction with various IPTG concentrations The arrow shows place of our overexpressed protein: 1. 22&deg;C 0.5 mM IPTG 2. 22&deg;C 0.1 mM IPTG 3. 22&deg;C 1 mM IPTG 4. Marker 5. negative control 6. 37&deg;C 0.1 mM IPTG 7. 37&deg;C 0.5 mM IPTG 8. 37&deg;C 1 mM IPTG

<img src="http://2008.igem.org/wiki/images/6/68/July_31_st_bis.jpg" width=350 /></a> Fig. 3. Pellets and supernatants from Z-alpha induction with various IPTG concentrations The arrow shows place of our overexpressed protein: 1. Marker 2. pellet negative control 3. supernatant negative control 4. pellet 37&deg;C 0.1 mM IPTG 5. supernatant 37&deg;C 0.1 mM IPTG 6. sonicate 22&deg;C 0.1 mM IPTG 7. sonicate 22&deg;C 0.5 mM IPTG 8. pellet 37&deg;C 0.5 mM IPTG 9. supernatant 37&deg;C 0.5 mM IPTG