Team:USyd-Australia/pUS204

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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 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
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

pUS204 Gene Cassette in pSB1C3 backbone