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− | <div style="width:75px;height: 15px;border: solid 1px gray;color: green;font-size:300%; line-height:40%;">••••</div>
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− | <p><i>UNIPV-Pavia iGEM 2010</i>
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− | <p><a class="part_link" href="https://parts.igem.org/wiki/index.php/Part:BBa_K300008">BBa_K300008</a> was used as a validation construct for this conditional replication origin, in order to test its capability to be propagated in pir+ or pir-116 strain and its inability to propagate in the other <i>E. coli</i> strains.
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− | </p><p>In particular, <a class="part_link" href="https://parts.igem.org/wiki/index.php/Part:BBa_K300008">BBa_K300008</a> was cut with XbaI-SpeI and the insert was isolated and purified from a 1% agarose gel. Then, it was self-ligated to generate a Cm-resistant R6K plasmid).
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− | </p><p>BW25141 (<a class="part_link" href="https://parts.igem.org/wiki/index.php/Part:BBa_K300984">BBa_K300984</a>) and BW23474 (<a class="part_link" href="https://parts.igem.org/wiki/index.php/Part:BBa_K300985">BBa_K300985</a>) were chosen as pir+ and pir-116 strains respectively, while DH5alpha (<a class="part_link" href="https://parts.igem.org/wiki/index.php/Part:BBa_V1001">BBa_V1001</a>), MC1061 (<a class="part_link" href="https://parts.igem.org/wiki/index.php/Part:BBa_K300078">BBa_K300078</a>) and MG1655 (<a class="part_link" href="https://parts.igem.org/wiki/index.php/Part:BBa_V1000">BBa_V1000</a>) were chosen as pir- strains.
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− | </p><p><br>
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− | All these strains were made competent following the commonly used CaCl2 method [Sambrook J, Fritsch EF, and Maniatis T (1989), Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.]. Then, a vial of 100 ul of competent cells was transformed with 2-4 ng of:
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− | </p>
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− | <ul><li>no DNA (negative control);</li>
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− | <li>a pSB*** series vector (positive control);</li>
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− | <li>self-ligated <a class="part_link" href="https://parts.igem.org/wiki/index.php/Part:BBa_K300008">BBa_K300008</a>.</li></ul>
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− | <p>and plated on LB+Cm at 34 ug/ml for high-copy plasmids, Cm at 12.5 ug/ml for medium/low copy plasmids and for the negative control strains transformed with the R6K plasmids.
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− | </p><p><br>
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− | The colonies were counted in each plate and the transformation efficiency was estimated in <b>[CFU/ug of DNA]</b> as:
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− | </p>
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− | <div align="center">
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− | <p><i>efficiency [CFU/ug of DNA]= # CFU * 1000 ng of DNA / amount of transformed DNA [ng]</i>
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− | </p>
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− | </div>
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− | <p>The results are shown here:
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− | </p>
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− | <td><b>Strain</b>
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− | </td>
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− | <td><b>Efficiency with no DNA</b>
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− | </td>
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− | <td><b>Efficiency with pSB*** (positive control)</b>
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− | </td>
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− | <td><b>Efficiency with the self-ligated <a class="part_link" href="https://parts.igem.org/wiki/index.php/Part:BBa_K300008">BBa_K300008</a> (R6K plasmid)</b>
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− | </td></tr>
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− | <tr>
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− | <td><a class="part_link" href="https://parts.igem.org/wiki/index.php/Part:BBa_K300084">BBa_K300084</a>
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− | </td>
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− | <td>0
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− | </td>
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− | <td>10^5
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− | </td>
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− | <td>10^5
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− | </td></tr>
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− | <tr>
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− | <td><a class="part_link" href="https://parts.igem.org/wiki/index.php/Part:BBa_K300085">BBa_K300085</a>
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− | </td>
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− | <td>0
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− | </td>
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− | <td>10^6
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− | </td>
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− | <td>10^6
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− | </td></tr>
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− | <tr>
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− | <td><a class="part_link" href="https://parts.igem.org/wiki/index.php/Part:BBa_V1001">BBa_V1001</a>
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− | </td>
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− | <td>0
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− | </td>
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− | <td>10^8
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− | </td>
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− | <td>0
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− | </td></tr>
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− | <tr>
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− | <td><a class="part_link" href="https://parts.igem.org/wiki/index.php/Part:BBa_K300078">BBa_K300078</a>
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− | </td>
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− | <td>0
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− | </td>
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− | <td>10^6
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− | </td>
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− | <td>0
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− | </td></tr>
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− | <tr>
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− | <td><a class="part_link" href="https://parts.igem.org/wiki/index.php/Part:BBa_V1000">BBa_V1000</a>
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− | </td>
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− | <td>0
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− | </td>
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− | <td>10^5
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− | </td>
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− | <td>0
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− | </td></tr></tbody></table>
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− | <p>These results show that <a class="part_link" href="https://parts.igem.org/wiki/index.php/Part:BBa_J61001">BBa_J61001</a> replication origin can be only propagated in pir+ and pir-116 strains (<a class="part_link" href="https://parts.igem.org/wiki/index.php/Part:BBa_K300084">BBa_K300084</a> and <a class="part_link" href="https://parts.igem.org/wiki/index.php/Part:BBa_K300085">BBa_K300085</a>), while the transformation of other strains with the R6K plasmid yielded no colonies after transformation.
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− | </p><p>Moreover, these results show that the R6K plasmid in pir+ and pir-116 strains was transformed with the same efficiency as the pSB*** positive control plasmid, demonstrating that the R6K origin doesn't give any handicap in plasmid transformation.
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− | </p>
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− | </td></tr></tbody></table>
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− | </html>
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