Phase 2: Plasmid Construction and Bacterial Transformations

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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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