Team:Warsaw/Calendar-Main/17 September 2008

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<h3>Competition tests</h3>
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<h3>'Hunter and prey' system tests: Competition tests</h3>
<h4>Piotr</h4>
<h4>Piotr</h4>
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<p><ol><li>Plasmid isolation from previous day's cultures.</li><li>Digest with SacI and BamHI.</li><li>Electrophoresis.</li></ol></p>
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<p><ol><li>Plasmid <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation>isolation</a> from previous day's cultures.</li>
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[photo of the gel is to be placed here]
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<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> with SacI and BamHI.</li><li>Electrophoresis (<a href="https://2008.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/17_September_2008#fig1">Fig. 1</a>).</li></ol></p>
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<p>Conclusion: cells with interracting protein survive competition!</p>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/9/98/Konkurencja1.jpg" width=180/></a>
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<var><b>Fig. 1.</b> Result of competition test:<br>
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1 and 5 - DNA ladder,<br>
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2 - Insert from isolated plasmid refers to OmpA_A_Alpha <br>(Z_Omega protein added to the medium),<br>
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3 - Insert from isolated plasmid refers to OmpA_A_Omega <br>(Z_Alpha protein added to the medium),<br>
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4 - Insert from isolated plasmid refers to OmpA_Z_Omega <br>(A_Alpha protein added to the medium).</var>
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<p>Conclusion: cells with interacting protein survive competition!</p>
<h3>MutD5 testing</h3>
<h3>MutD5 testing</h3>
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<h4>Michał K.</h4>
<h4>Michał K.</h4>
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<ol><li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on pACYC177+OmpA_omega_deltaA_alpha plasmid using  
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<ol><li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>pACYC177+OmpA_omega_&Delta;A_alpha</a> plasmid using  
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL_BNXNE">AL_BNXNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#APSacSpe">APSacSpe</a>
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL_BNXNE">AL_BNXNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#APSacSpe">APSacSpe</a>
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  primers (elongation length 45s) to obtain deltaA fragment. </li>
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  primers (elongation length 45s) to obtain &Delta;A fragment. </li>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on pACYC177+OmpA_omega_deltaA_alpha plasmid using  
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>pACYC177+OmpA_omega_&Delta;A_alpha</a> plasmid using  
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinLSXNE">LinLSXNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlfaPSpe">AlfaPSpe</a>
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinLSXNE">LinLSXNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlfaPSpe">AlfaPSpe</a>
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primers (elongation length 60s) to obtain link_omega fragment. </li>
primers (elongation length 60s) to obtain link_omega fragment. </li>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on pACYC177+OmpA_omega_deltaA_alpha plasmid using
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>pACYC177+OmpA_omega_&Delta;A_alpha</a> plasmid using
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmpLNXNE">OmpLNXNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinP_BS">LinP_BS</a>
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmpLNXNE">OmpLNXNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinP_BS">LinP_BS</a>
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primers (elongation length 90s) to obtain Omp_omega fragment. </li>
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primers (elongation length 90s) to obtain OmpA_omega fragment. </li>
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<p>Each reaction was carried out with 25 cycles and in temperature gradient from 55 to 75 &deg;C.</p>
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<p>Each reaction was carried out with 25 cycles and in temperature gradient from 55 to 75 &deg;C (four reactions).</p>
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<li> Gel electrophoresis of PCR products (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/17_September_2008#fig2">Fig. 2.</a>).</li>
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</ol>
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<a name="fig2"><img src="https://static.igem.org/mediawiki/2008/9/9e/Grad_17_09.jpg" width=300/></a><var><b>Fig. 2.</b> Gradient PCR (temperatures:55-75 &deg;C)<br>
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1. Marker<br>
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2. 55 &deg;C deltaA<br>
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3. 60 &deg;C deltaA<br>
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4. 65 &deg;C deltaA<br>
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5. 70 &deg;C deltaA<br>
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6. 55 &deg;C link_omega<br>
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7. 60 &deg;C link_omega<br>
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8. 65 &deg;C link_omega<br>
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9. 70 &deg;C link_omega<br>
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10. 55 &deg;C link_alpha<br>
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11. 60 &deg;C link_alpha<br>
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12. 65 &deg;C link_alpha<br>
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13. 70 &deg;C link_alpha<br>
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14. 55 &deg;C OmpA_omega<br>
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15. 60 &deg;C OmpA_omega<br>
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16. 65 &deg;C OmpA_omega<br>
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17. 70 &deg;C OmpA_omega<br></var>
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Latest revision as of 15:15, 29 October 2008

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'Hunter and prey' system tests: Competition tests

Piotr

  1. Plasmid isolation from previous day's cultures.
  2. Digest with SacI and BamHI.
  3. Electrophoresis (Fig. 1).

Fig. 1. Result of competition test:
1 and 5 - DNA ladder,
2 - Insert from isolated plasmid refers to OmpA_A_Alpha
(Z_Omega protein added to the medium),
3 - Insert from isolated plasmid refers to OmpA_A_Omega
(Z_Alpha protein added to the medium),
4 - Insert from isolated plasmid refers to OmpA_Z_Omega
(A_Alpha protein added to the medium).

Conclusion: cells with interacting protein survive competition!

MutD5 testing

Emilia

Inoculation of MutD5 into medium with tetracycline.

Optimisation of primers for preparation of parts

Michał K.

  1. PCR on pACYC177+OmpA_omega_ΔA_alpha plasmid using AL_BNXNE and APSacSpe primers (elongation length 45s) to obtain ΔA fragment.
  2. PCR on pACYC177+OmpA_omega_ΔA_alpha plasmid using LinLSXNE and AlfaPSpe primers (elongation length 60s) to obtain link_alpha fragment.
  3. PCR on pACYC177 + OmpA_omega plasmid using LinLSXNE and OmegPSpe primers (elongation length 60s) to obtain link_omega fragment.
  4. PCR on pACYC177+OmpA_omega_ΔA_alpha plasmid using OmpLNXNE and LinP_BS primers (elongation length 90s) to obtain OmpA_omega fragment.

    5. Each reaction was carried out with 25 cycles and in temperature gradient from 55 to 75 °C (four reactions).

  5. Gel electrophoresis of PCR products (Fig. 2.).
Fig. 2. Gradient PCR (temperatures:55-75 °C)
1. Marker
2. 55 °C deltaA
3. 60 °C deltaA
4. 65 °C deltaA
5. 70 °C deltaA
6. 55 °C link_omega
7. 60 °C link_omega
8. 65 °C link_omega
9. 70 °C link_omega
10. 55 °C link_alpha
11. 60 °C link_alpha
12. 65 °C link_alpha
13. 70 °C link_alpha
14. 55 °C OmpA_omega
15. 60 °C OmpA_omega
16. 65 °C OmpA_omega
17. 70 °C OmpA_omega


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