Team:Warsaw/Calendar-Main/30 July 2008

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<h3>Purification of proteins: Z-alpha and Z-omega</h3><h4>Piotr, Emilia</h4>
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<h3>Purification of proteins: Z-alpha and Z-omega</h3><h4>Piotr, Emilia, Weronika</h4>
<p><ol><li><a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BZ-alpha>pET15b+Z_alpha</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2Bhis%2BZ%2Bomega>pET15b+Z_omega</a> in <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#rosetta">Rosetta</a> strain from overnight culture inoculated in fresh LB and cultured until OD=0,5.</li>
<p><ol><li><a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BZ-alpha>pET15b+Z_alpha</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2Bhis%2BZ%2Bomega>pET15b+Z_omega</a> in <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#rosetta">Rosetta</a> strain from overnight culture inoculated in fresh LB and cultured until OD=0,5.</li>
<li>Cultures induced with different concentrations of IPTG in 22&deg;C and 37&deg;C.</li>
<li>Cultures induced with different concentrations of IPTG in 22&deg;C and 37&deg;C.</li>
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<h3> Cloning of truncated fragment of protein A </h3>
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<h3> Cloning of truncated fragment of protein A (&Delta;A)</h3>
<h4>Michał K.</h4>
<h4>Michał K.</h4>
<ol><li>Optimization of <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> to obtain truncated fragment of protein A DNA.<br>
<ol><li>Optimization of <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> to obtain truncated fragment of protein A DNA.<br>
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Elongation time: 30s <br>
Elongation time: 30s <br>
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- Optimization of annealing temperature (gradient from 55&deg;C to 75&deg;C) )<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/30_July_2008#fig1">Fig. 1.</a>).<br>  
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- Optimization of annealing temperature (gradient from 55&deg;C to 75&deg;C). <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/30_July_2008#fig1">Fig. 1</a>.<br>  
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- Optimization of number of cycles(15, 20, 25, 30, 35) (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/30_July_2008#fig2">Fig. 2.</a>).</li>
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- Optimization of number of cycles(15, 20, 25, 30, 35). <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/30_July_2008#fig2">Fig. 2</a>.</li>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> to obtain truncated A protein DNA fragment. <br>
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> to obtain truncated A protein DNA fragment. <br>
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20 cycles </li>
20 cycles </li>
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<li>Gel electrophoresis and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of 250 bp band. </li>
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<li>Gel electrophoresis and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of 250 bp band. <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/30_July_2008#fig3">Fig. 3</a>. </li>
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<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> of isolated PCR product, <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega>pACYC177+OmpA_omega</a> with NotI and SacI (BamHI buffer). pACYC177 vectors were also <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">dephosphorylated</a>. </li>
<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> of isolated PCR product, <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega>pACYC177+OmpA_omega</a> with NotI and SacI (BamHI buffer). pACYC177 vectors were also <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">dephosphorylated</a>. </li>
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_purification_after_enzymatic_reaction">Clean-up</a> of digest reaction. </li>
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_purification_after_enzymatic_reaction">Clean-up</a> of digest reaction. </li>
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<li>Overnight <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">ligation</a>: pACYC177+OmpA_alpha + &Delta;A and pACYC177+OmpA_omega + &Delta;A.</li></ol>
<li>Overnight <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">ligation</a>: pACYC177+OmpA_alpha + &Delta;A and pACYC177+OmpA_omega + &Delta;A.</li></ol>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/3/32/Nazwa_pliku.jpg" width=300/></a> <var><b>Fig. 1.</b> Gradient PCR to obtain shortened protein A (temperatures: 55-75&deg;C)<br>  
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/3/32/Nazwa_pliku.jpg" width=300/></a> <var><b>Fig. 1.</b> Gradient PCR to obtain truncated protein A (temperatures: 55-75&deg;C)<br>  
1. Marker<br>
1. Marker<br>
2. 50&deg;C<br>
2. 50&deg;C<br>
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7. 75&deg;C<br></var>
7. 75&deg;C<br></var>
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<a name="fig2"><img src="https://static.igem.org/mediawiki/2008/7/75/Plik.jpg" width=300/></a> <var><b>Fig. 2. </b>PCR to obtain shortened protein A (various number of cycles)<br>  
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<a name="fig2"><img src="https://static.igem.org/mediawiki/2008/7/75/Plik.jpg" width=300/></a> <var><b>Fig. 2. </b>PCR to obtain truncated protein A (various number of cycles)<br>  
1. Marker<br>
1. Marker<br>
2. 15 cycles<br>
2. 15 cycles<br>
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6. 35 cycles<br></var>
6. 35 cycles<br></var>
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<a name="fig3"><img src="https://static.igem.org/mediawiki/2008/c/c4/Go_26_08_2008.jpg" width=300/></a><var><b>Fig. 3.</b> PCR amplified truncated protein A<br>
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1. Marker<br>
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2. PCR product (deltaA), temperature of annealing = 60&deg;C, 20 cycles<br></var>
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Purification of proteins: Z-alpha and Z-omega

Piotr, Emilia, Weronika

  1. pET15b+Z_alpha and pET15b+Z_omega in Rosetta strain from overnight culture inoculated in fresh LB and cultured until OD=0,5.
  2. Cultures induced with different concentrations of IPTG in 22°C and 37°C.
  3. Samples were collected twice: after 3h and next day (samples were centrifuged and frozen).

Cloning of omega_ΔA DNA fragment to pACYC177+OmpA_alpha

Michał K.

Separate transformant colonies (tranformation of ligation of pACYC177+OmpA_alpha and omega_ΔA from previous day) inoculated to liquid LB with kanamycin.

Cloning of truncated fragment of protein A (ΔA)

Michał K.

  1. Optimization of PCR to obtain truncated fragment of protein A DNA.
    Primers: AL+SacI AP+NotI
    Template DNA: pDRIVE-TapTag
    Elongation time: 30s
    - Optimization of annealing temperature (gradient from 55°C to 75°C). Fig. 1.
    - Optimization of number of cycles(15, 20, 25, 30, 35). Fig. 2.
  2. PCR to obtain truncated A protein DNA fragment.
    Primers: AL+SacI AP+NotI
    Template DNA: pDRIVE-TapTag
    Elongation time: 30s
    Annealing temperature: 60°C
    20 cycles
  3. Gel electrophoresis and gel-out of 250 bp band. Fig. 3.
  4. Digest of isolated PCR product, pACYC177+OmpA_alpha and pACYC177+OmpA_omega with NotI and SacI (BamHI buffer). pACYC177 vectors were also dephosphorylated.
  5. Clean-up of digest reaction.
  6. Gel electrophoresis for estimation of DNA concentration.
  7. Overnight ligation: pACYC177+OmpA_alpha + ΔA and pACYC177+OmpA_omega + ΔA.
Fig. 1. Gradient PCR to obtain truncated protein A (temperatures: 55-75°C)
1. Marker
2. 50°C
3. 55°C
4. 60°C
5. 65°C
6. 70°C
7. 75°C
Fig. 2. PCR to obtain truncated protein A (various number of cycles)
1. Marker
2. 15 cycles
3. 20 cycles
4. 25 cycles
5. 30 cycles
6. 35 cycles
Fig. 3. PCR amplified truncated protein A
1. Marker
2. PCR product (deltaA), temperature of annealing = 60°C, 20 cycles