Phase 2: Plasmid Construction and Bacterial Transformations
From 2006.igem.org
1. Isolated ptetRBS and pSB1A3 plasmids from BioBricks
- Attached tet promoter with Ribosome Binding Site to GFP gene, transformed XL-1 E. coli and observed positive expression
2. Ligated ComQX/P/A and Tsr fragments from Phase 1 into pSB1A3 plasmids
- Cut at EcoRI, XbaI, and PstI restriction sites (cutting at XbaI site reduces background)
- pSB1A3 contains amp resistance
- Concentration of digested vector and inserts determined. Ligations were set up for between 40:150 to 40ng:300ng ratio vector:insert
3. Bacterial transformation
- Used E.coli strain RP8611 (null for all MCP receptors, provided by John S. Parkinson, University of Utah)
- Verified transformation for all fragments and sequenced genes (results positive in all cases).
4. Significance:
- We now have all the fundamental components for the project ahead
- Can proceed in constructing the hybrid quorumtaxis receptor using ComP and Tsr genes
- Will need to perform quick-change mutagenesis for ComA to remove internal SpeI site
