Team:Warsaw/Calendar-Main/31 July 2008

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<center><h4><p>1. Optimization of PCR to obtain truncated fragment of protein A DNA </center>
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<h3> Cloning of truncated fragment of protein A (&Delta;A)</h3>  
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</h4>
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<h4> Michał K.</h4>
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Primers: <html>
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<p><ol>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+SacI ">AL+SacI</a>
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<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#top">TOP10</a> strain with ligation products (<a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-deltaA-alpha>pACYC177+OmpA_alpha + &Delta;A</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BOmpA-deltaA-omega>pACYC177+OmpA_omega + &Delta;A</a>). </li>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> </html>
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<li> Transformants plating on LB + kanamycin. </li></ol></p>
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Elongation time: 30s <br>
 
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- Optimization of annealing temperature (gradient from 55&deg;C to 75&deg;C)<br>
 
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- Optimization of number of cycles(15, 20, 25, 30, 35)<br>
 
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2. PCR to obtain truncated A protein DNA fragment <br>
 
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<p>Primers:<html>
 
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+SacI ">AL+SacI</a>
 
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a></html></p>
 
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Elongation time: 30s <br>
 
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Annealing temperature: 60&deg;C <br>
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<h3>Cloning of omega_&Delta;A DNA fragment to <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</a></h3><h4>Michał K.</h4>
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20 cycles <br>
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<p>
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<ol><li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation of plasmids</a> from cultures inocluated on <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/31_July_2008">previous day</a>. </li>
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<li> Control <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">digest</a> of isolated plasmids with BamHI and SacI (BamHI buffer). </li><li> Gel electrophoresis - proper clones founded (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/31_July_2008#fig1">Fig. 1.</a>). </li></ol>
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</p>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/2/2e/31_july.jpg" width=250/></a> <var><b>Fig. 1. </b>Control SacI/BamHI digests of isolated plasmids<br>
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1-4. digested plasmids pACYC177 + OmpA-omega-&Delta;A-alpha<br>
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5. Marker<br></var>
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3. Gel electrophoresis and isolation of 250 bp band. <br>
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<h3>Purification of proteins: Z-alpha and Z-omega</h3><h4>Piotr, Emilia</h4>
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4. Digest of isolated PCR product, pACYC177+OmpA_alpha and pACYC177+OmpA_omega with NotI and SacI. <br>
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<p><ol><li>Samples were resuspended in PBS, sonicated and centrifuged.</li>
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5. Clean-up of digestion reaction. <br>
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<li>Lysis buffer was added separately to pellet and supernatant, then samples were boiled for 10 min. </li>
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6. Gel electrophoresis for estimation of DNA concentration. <br>
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<li>Lysates loaded on 12% poliacrylamide gel (amount relating to 100 μl of OD=1.0 culture).</li>
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7. Overnight ligation: pACYC177+OmpA_alpha + deltaA and pACYC177+OmpA_omega + deltaA.
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<li>Gel stained with Coomassie Blue. Optimal induction conditions chosen (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/31_July_2008#fig2">Fig. 2.</a> and <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/31_July_2008#fig3">Fig. 3.</a>).</li></ol>
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<a name="fig2"><img src="https://static.igem.org/mediawiki/2008/e/e7/July_31_st.jpg" width=350 /></a><var><b>Fig. 2.</b> Pellets from Z-omega induction with various IPTG concentrations<br>
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The arrow shows place of our overexpressed protein:<br>
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1. 22&deg;C 0.5 mM IPTG<br>
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2. 22&deg;C 0.1 mM IPTG<br>
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3. 22&deg;C 1 mM IPTG<br>
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4. Marker<br>
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5. negative control<br>
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6. 37&deg;C 0.1 mM IPTG<br>
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7. 37&deg;C 0.5 mM IPTG<br>
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8. 37&deg;C 1 mM IPTG<br></var>
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<a name="fig3"><img src="https://static.igem.org/mediawiki/2008/6/68/July_31_st_bis.jpg" width=350 /></a><var><b>Fig. 3. </b>Pellets and supernatants from Z-alpha induction with various IPTG concentrations<br>
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The arrow shows place of our overexpressed protein:<br>
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1. Marker<br>
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2. pellet negative control<br>
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3. supernatant negative control<br>
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4. pellet 37&deg;C 0.1 mM IPTG <br>
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5. supernatant 37&deg;C 0.1 mM IPTG<br>
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6. sonicate 22&deg;C 0.1 mM IPTG<br>
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7. sonicate 22&deg;C 0.5 mM IPTG<br>
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8. pellet 37&deg;C 0.5 mM IPTG<br>
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9. supernatant 37&deg;C 0.5 mM IPTG<br></var>
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<center><h4>Checking if degradation of fusion with OmpA is a result of Top10 proteases' activity (lon, iompt)</center>Piotr:</h4>
 
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Inoculation of OmpA_omega_A_alpha and OmpA_A_alpha with and without inductor (0,5IPTG) in <i>E. coli</i> Rosetta strain.
 
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Cloning of truncated fragment of protein A (ΔA)

Michał K.

  1. Transformation of E. coli TOP10 strain with ligation products (pACYC177+OmpA_alpha + ΔA and pACYC177+OmpA_omega + ΔA).
  2. Transformants plating on LB + kanamycin.

Cloning of omega_ΔA DNA fragment to pACYC177+OmpA_alpha

Michał K.

  1. Isolation of plasmids from cultures inocluated on previous day.
  2. Control digest of isolated plasmids with BamHI and SacI (BamHI buffer).
  3. Gel electrophoresis - proper clones founded (Fig. 1.).

Fig. 1. Control SacI/BamHI digests of isolated plasmids
1-4. digested plasmids pACYC177 + OmpA-omega-ΔA-alpha
5. Marker

Purification of proteins: Z-alpha and Z-omega

Piotr, Emilia

  1. Samples were resuspended in PBS, sonicated and centrifuged.
  2. Lysis buffer was added separately to pellet and supernatant, then samples were boiled for 10 min.
  3. Lysates loaded on 12% poliacrylamide gel (amount relating to 100 μl of OD=1.0 culture).
  4. Gel stained with Coomassie Blue. Optimal induction conditions chosen (Fig. 2. and Fig. 3.).
Fig. 2. Pellets from Z-omega induction with various IPTG concentrations
The arrow shows place of our overexpressed protein:
1. 22°C 0.5 mM IPTG
2. 22°C 0.1 mM IPTG
3. 22°C 1 mM IPTG
4. Marker
5. negative control
6. 37°C 0.1 mM IPTG
7. 37°C 0.5 mM IPTG
8. 37°C 1 mM IPTG
 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°C 0.1 mM IPTG
5. supernatant 37°C 0.1 mM IPTG
6. sonicate 22°C 0.1 mM IPTG
7. sonicate 22°C 0.5 mM IPTG
8. pellet 37°C 0.5 mM IPTG
9. supernatant 37°C 0.5 mM IPTG

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