We are in Week 9 of the iGEM project
Purification of LuxR
Two DNA constructs are needed for the purification of LuxR
- one containing the His-tag
- one over expressing LuxR
Currently we only have the overexpressing LuxR plasmid. From literature, we have found out that the LuxR needs AHL to fold correctly.
Two purification methods are to be pursued (we have discussed this with Frank):
- 1st: run the LuxR through a ion exchange column in its already folded state
- 2nd: purify from an inclusion bodies
We are going to grow the cell and test to see which method are more suitable, the first relies upon soluble protein the second relies upon insoluble proteins. Growing up of cells will take 2 days, and testing will be carried out thereafter.
Further Testing of [AHL]
- Concentrations of 3nM,5nM and 7nM of [AHL] will be tested tomorrow.
- Specification of lowest threshold of detection has been changed to 50nM, but since 10nM of [AHL] gives a significant response, we will continue testing the range 1-10nM to find the sensitivity of the system.
GFP Calibration Curve
- The GFP calibration curve has been constructed (a linear relationship between fluorescence and concentration of GFP exists within our testing range)
- There is quite alot of variability, however we feel that we can use it as it is.
- We also carried out a quick test about the visibility of GFP with a haldheld UV light, and 1uM was found to have a clearly visible fluorescence.
Cell By Date
- Data generated from staggering the experiments so as to obtain more data during the middle of the 24 hours period was found to be extremely unpredictable, and did not tally with the results of the initial samples done for the first and last hours of the 24 hour period.
- We are looking into the variability of fluorescence from staggered readings; the possible causes of the varibility and how we can go about solving the problem.