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Team:USyd-Australia/pUS204
From 2014.igem.org
(Difference between revisions)
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The BioBrick parts were ordered as a synthetic gBlock from IDT, along with primers to amplify the entire gBlock by PCR. | The BioBrick parts were ordered as a synthetic gBlock from IDT, along with primers to amplify the entire gBlock by PCR. | ||
| - | pSB1C3 linearised backbone was amplified by PCR using new primers to re-introduce the full biobrick prefix and suffix. The two parts were joined by restriction-ligation using EcoRI and PstI. The components | + | pSB1C3 linearised backbone was amplified by PCR using new primers to re-introduce the full biobrick prefix and suffix. The two parts were joined by restriction-ligation using EcoRI and PstI. The components were assembled as a circular plasmid in the order: pSB1C3 → AttC → aeBlue → aacC1 GmR→ pSB1C3. To retrieve functional cassettes, PCR primers containing compatible but non-identical restriction enzyme sites (XbaI and SpeI) at the end. From PCR products, circular cassettes were generated using Enzymatic Ligation Assisted by Nucleases (ELAN) via the XbaI and SpeI sites. |
</div> | </div> | ||
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</ul> | </ul> | ||
<li>Primers<ul> | <li>Primers<ul> | ||
| - | <li>pSB1C3 linearisation primers containing edited biobrick prefix and suffix, | + | <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 | ||
<li>2 sets of junction primers</ul> | <li>2 sets of junction primers</ul> | ||
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<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 | + | Part 1: Design and order gBlock for aeBlue-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: Preparation of pSB1C3 backbone by XbaI digestion</br> | Part 3: Preparation of pSB1C3 backbone by XbaI digestion</br> | ||
Revision as of 10:08, 17 October 2014
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pUS204 Gene Cassette in pSB1C3 backbone
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.
Approach
The BioBrick parts were ordered as a synthetic gBlock from IDT, along with primers to amplify the entire gBlock by PCR.
pSB1C3 linearised backbone was amplified by PCR using new primers to re-introduce the full biobrick prefix and suffix. The two parts were joined by restriction-ligation using EcoRI and PstI. The components were assembled as a circular plasmid in the order: pSB1C3 → AttC → aeBlue → aacC1 GmR→ pSB1C3. To retrieve functional cassettes, PCR primers containing compatible but non-identical restriction enzyme sites (XbaI and SpeI) at the end. From PCR products, circular cassettes were generated using Enzymatic Ligation Assisted by Nucleases (ELAN) via the XbaI and SpeI sites.
Materials
Method
Part 1: Design and order gBlock for aeBlue-AttC construct
Part 2: Perform PCR on pSB1C3 linearised backbone (kit
Part 3: Preparation of pSB1C3 backbone by XbaI digestion
Part 4: Gibson Assembly of the three components.
Validation
With thanks to:
