Team:USyd-Australia/pUS204
From 2014.igem.org
Line 6: | Line 6: | ||
<h2>pUS204 Gene Cassette in pSB1C3 backbone</h2> | <h2>pUS204 Gene Cassette in pSB1C3 backbone</h2> | ||
- | + | <img src="https://static.igem.org/mediawiki/2014/1/1c/USyd-Australia_pUS204_Map.png" align="right" width="60%"> | |
<h3 onclick="toggle_visibility('Aim');">Aim</h3> | <h3 onclick="toggle_visibility('Aim');">Aim</h3> | ||
<div id="Aim"> | <div id="Aim"> | ||
- | |||
To construct a reporter gene cassette consisting of aeBlue reporter and gentamicin resistance selective marker, and attC site. The cassette must be cloned into standard iGEM pSB1C3 backbone with all sequences void of forbidden restriction sites as per iGEM requirements. The cassette must be easily recovered as a circular product from the backbone for further work. | To construct a reporter gene cassette consisting of aeBlue reporter and gentamicin resistance selective marker, and attC site. The cassette must be cloned into standard iGEM pSB1C3 backbone with all sequences void of forbidden restriction sites as per iGEM requirements. The cassette must be easily recovered as a circular product from the backbone for further work. | ||
Line 32: | Line 31: | ||
<li>Primers<ul> | <li>Primers<ul> | ||
<li>pSB1C3 linearisation primers containing edited biobrick prefix and suffix, <a href="https://2014.igem.org/Team:USyd-Australia/Notebook/Primers#iGEM1417">iGEM1417</a> and <a href="https://2014.igem.org/Team:USyd-Australia/Notebook/Primers#iGEM1418">iGEM1418</a> | <li>pSB1C3 linearisation primers containing edited biobrick prefix and suffix, <a href="https://2014.igem.org/Team:USyd-Australia/Notebook/Primers#iGEM1417">iGEM1417</a> and <a href="https://2014.igem.org/Team:USyd-Australia/Notebook/Primers#iGEM1418">iGEM1418</a> | ||
- | <li>aeBlue-aacC1 GmR-AttC gblock linearisation primers | + | <li>aeBlue-aacC1 GmR-AttC gblock linearisation primers <a href="https://2014.igem.org/Team:USyd-Australia/Notebook/Primers#iGEM1419">iGEM1419</a> and <a href="https://2014.igem.org/Team:USyd-Australia/Notebook/Primers#iGEM1420">iGEM1420</a> |
- | <li> | + | <li>Junction primers <a href="https://2014.igem.org/Team:USyd-Australia/Notebook/Primers#iGEM1407">iGEM1407</a> and <a href="https://2014.igem.org/Team:USyd-Australia/Notebook/Primers#iGEM1408">iGEM1408</a></ul> |
</ul> | </ul> | ||
</div> | </div> | ||
Line 39: | Line 38: | ||
<h3 onclick="toggle_visibility('Method');">Method</h3> | <h3 onclick="toggle_visibility('Method');">Method</h3> | ||
<div id="Method"> | <div id="Method"> | ||
- | Part 1: Design and order gBlock for aeBlue-AttC construct</br> | + | Part 1: Design and order gBlock for aeBlue-GmR-AttC construct</br> |
- | Part 2: Perform PCR on pSB1C3 linearised backbone (kit</br> | + | Part 2: Perform PCR on pSB1C3 linearised backbone (kit)</br> |
- | Part 3: | + | Part 3: Perform PCR on aeBlue-GmR-AttC gBlock</br> |
- | Part 4: | + | Part 4: Clone by digestion-ligation via EcoRI and PstI sites |
</div> | </div> | ||
+ | |||
+ | <h3>Results</h3> | ||
+ | |||
+ | <p>PCR amplified gBlock was <a href="https://2014.igem.org/Team:USyd-Australia/Project/Protocols#restrict">cut</a> with EcoRI and PstI, and <a href="https://2014.igem.org/Team:USyd-Australia/Project/Protocols#ligation">ligated</a> into similarly cut linearised pSB1C3 backbone. Ligation mixtures were transformed by heat shock into Top10 competent <i>E. coli</i>, and plated onto Chloramphenicol selective LB agar. Chloramphenicol resistant colonies were picked and subjected to junction PCRs of direct colonies using primers <a href="https://2014.igem.org/Team:USyd-Australia/Notebook/Primers#iGEM1407">iGEM1407</a> and <a href="https://2014.igem.org/Team:USyd-Australia/Notebook/Primers#iGEM1408">iGEM1408</a></ul>. 26/27 of the colonies screened were positive at the expected amplicon size of 449bp. Two clones, 9 and 17, were selected from the patch plate generated before <a href="https://2014.igem.org/Team:USyd-Australia/Project/Protocols#PCR">colony PCR</a>, and plasmids extracted via <a href="https://2014.igem.org/Team:USyd-Australia/Project/Protocols#plasmid>plasmid mini prep</a> | ||
<h3 onclick="toggle_visibility('Validation');">Validation</h3> | <h3 onclick="toggle_visibility('Validation');">Validation</h3> | ||
<div id="Validation"> | <div id="Validation"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/c/c4/USyd-Australia_Integron_Design-Produce_Cassettes.png" width="300px" align="left"> | ||
+ | <p>To validate the function of the cassette, PCR products of the aeBlue-GmR gBlock were subjected to <a href="https://2014.igem.org/Team:USyd-Australia/Project/Protocols#ELAN"> ELAN</a> to produce circular cassettes. | ||
</div> | </div> |
Revision as of 03:45, 18 October 2014
pUS204 Gene Cassette in pSB1C3 backbone
Aim
Approach
Materials
Method
Results
PCR amplified gBlock was cut with EcoRI and PstI, and ligated into similarly cut linearised pSB1C3 backbone. Ligation mixtures were transformed by heat shock into Top10 competent E. coli, and plated onto Chloramphenicol selective LB agar. Chloramphenicol resistant colonies were picked and subjected to junction PCRs of direct colonies using primers iGEM1407 and iGEM1408. 26/27 of the colonies screened were positive at the expected amplicon size of 449bp. Two clones, 9 and 17, were selected from the patch plate generated before colony PCR, and plasmids extracted via Validation
To validate the function of the cassette, PCR products of the aeBlue-GmR gBlock were subjected to ELAN to produce circular cassettes.