Team:BioBricks/Demonstrate
To test if the daisy chain performed the way that we expected we built four different architectures with one variable in each architecture.
One variable that we knew could affect the way the operon functioned was the ribosome binding site that was inserted into the end of the first gene. Using EMOPAC’s calculations we identified three sequences that supposedly expressed RFP at 80%, 40% and 15% of total expression. These daisy chains were named DC CFP RFP 80, DC CFP RFP 40, and DC CFP RFP 15.
Another variable that we knew could affect the expression levels was the order of the genes, so we created a daisy chain that had RFP as the first gene with an inserted RBS, and followed by CFP. We called this daisy chain: DC RFP CFP.
All of the four daisy chains had the same overall architecture: Ptrc* promoter, BCD 5 high, and a double terminator.
With these daisy chains built we used a fluorescence test to see if the CFP and RFP were visible. Without quantification, both fluorescent proteins were visible through fluorescent microscopy in a saturated growth stage.
