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

  • Used SpeI and XbaI restriction sites
  • Verified transformation for all fragments and sequenced genes (results positive in all cases).



3. Significance:

  • We now have all the fundamental components for the project ahead
  • Can proceed in constructing the chimera using ComP and Tsr gene fragments
  • Will need to perform quick-change mutagenesis for ComA to remove internal SpeI site
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