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

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