Figure 1: The robotic liquid handling system above was used in our testing protocol to dispense the autoinducer into our well plates.

The characterization of our parts was a long and detailed process involving weeks of testing and analysis. However, the general process was similar for most of our samples:

1. The night before testing, grow overnight cultures of all samples and controls in M9 Media.
2. In the morning, dilute all the cultures to an optical density of 0.05.
3. Let grow for approximately 3-4 hours, monitoring the OD to make sure it does not grow above 0.5.
4. When the OD of the samples reaches 0.5, distribute the samples into a 96 or 384 well plate.
5. While waiting for the cells to grow, dilute the autoinducers such that they will have the desired concentration when added to the well plate. Our concentration range was generally on the order of 1uM.
6. Simultaneously induce with PAI1 and/or PAI2 using a robotic liquid handling system like the one pictured above.
7. Put the plate in a Synergy fluorescence plate reader, take regular measurements of OD and fluorescence. For GFP, we used an excitation frequency of 485nm and an emission frequency of 528nm. For RFP, we used an excitation frequency of 580nm and an emission frequency of 610nm.