From 2014.igem.org
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
- DNA
- pSB1C3, linearised - by PCR
- aeBlue-aacC1 GmR-AttC - gBlock order from IDT
- Primers
Method
Part 1: Design and order gBlock for aeBlue-GmR-AttC construct
Part 2: Perform PCR on pSB1C3 linearised backbone (kit)
Part 3: Perform PCR on aeBlue-GmR-AttC gBlock
Part 4: Clone by digestion-ligation via EcoRI and PstI sites
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.
With thanks to:
"
