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

• 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