pUS204 Gene Cassette in pSB1C3 backbone |
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 |
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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.
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 |
- DNA
- pSB1C3, linearised - by PCR
- aeBlue-aacC1 GmR-AttC - gBlock order from IDT
- Primers
- pSB1C3 linearisation primers containing edited biobrick prefix and suffix
- aeBlue-aacC1 GmR-AttC gblock linearisation primers
- 2 sets of junction primers
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Method outline |
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Part 1: Design and order gBlock for aeBlue-AttC construct AND primers for obtaining aacC1 GmR from pUCP24.
Part 2: Perform PCR on pUCP24 whole genome to obtain aacC1 GmR gene (with Gibson ends overlaps)
Part 3: Preparation of pSB1C3 backbone by XbaI digestion “Note: The iGEM Prefix is required to end in an AGAG, or just AG if the first three bases of the insert are ATG. The first three bases of the aacC1 RBS are ATG, so we may have to do an XbaI digest on the pSB1C3 backbone before Gibson assembly. Alternatively, we can just insert an extra base between the annealing section and Gibson end and keep it as AGAG.”
Part 4: Gibson Assembly of the three components. |
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