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Team:USyd-Australia/pUS204
From 2014.igem.org
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<tr><td colspan="2"><h2>pUS204 Gene Cassette in pSB1C3 backbone</h2></td> | <tr><td colspan="2"><h2>pUS204 Gene Cassette in pSB1C3 backbone</h2></td> | ||
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<tr><td width="320px"><img src="https://static.igem.org/mediawiki/2014/c/c4/USyd-Australia_Integron_Design-Produce_Cassettes.png" width="300px"> | <tr><td width="320px"><img src="https://static.igem.org/mediawiki/2014/c/c4/USyd-Australia_Integron_Design-Produce_Cassettes.png" width="300px"> | ||
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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.</td> </tr> | 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.</td> </tr> | ||
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<tr><td><h3>Approach</h3></td></tr> | <tr><td><h3>Approach</h3></td></tr> | ||
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<tr><td colspan="2"><h3>Method outline</h3></td></tr> | <tr><td colspan="2"><h3>Method outline</h3></td></tr> | ||
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| - | Part 1: Design and order gBlock for aeBlue-AttC construct</br> | + | Part 1: Design and order gBlock for aeBlue-AttC construct <a href="https://2014.igem.org/Team:USyd-Australia/Notebook/Primers#iGEM10">iGEM10</a></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 09:36, 17 October 2014
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pUS204 Gene Cassette in pSB1C3 backbone |
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Aim | |
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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 will be assembled as a circular plasmid in the order: pSB1C3 → aacC1 GmR → aeBlue → AttC → pSB1C3 | |
Materials | |
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Method outline | |
| Part 1: Design and order gBlock for aeBlue-AttC construct iGEM10 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:
