IGEM:Brown/2007/Sensor/AHL results on T9002
We had a lot of success working with AHL and the T9002 construct.
We used Imperial's AHL concentrations as starting points. We used 100 ul of cultured T9002 in LB to create:
0 nM AHL
1 nM AHL
100 nM AHL
and 10,000 nM AHL solution
Our 0 AHL sample was used to show the amount of leakyness within our cell. Some GFP was expressed, about 2000 RFU. (How much does just LB express?)
We used the Nanodrop Spectrophotometer and fluorospec to analyze samples.
1. Each sample was cultured in LB, which we expected to have a linear response to AHL (this is what Imperial's observations showed). From the data, it appears that this held true. We will be trying a culture of the cells in M9, for which Imperial's results show a "switch response".
2. We also found that cell concentrations could vary widely between samples. This would result in higher RFU values. Therefore, we compensated by dividing RFU by concentration. This required taking both a concentration value and RFU test for each culture.
However, since the cultures were each grown under identical conditions for identical periods, VORTEXING the cells made each sample homogeneous.