Phase 1 - Intial Testing
Intialy testing of various simlpe constructs to confirm there is gene expression in vitro and in veso, the simlpe constructs are:
|Test Constructs||Device Implemented|
|Constitutive E.coli Promoter||Cell by Date|
|Constitutive T7 Promoter||Cell by Date|
|Inducible E.coli Promoter||Cell by Date|
|Inducible E.coli Promoter||Biofilm|
Firstly we will test the promoters in vitro system. We will be simply testing whether the promoters actually work so we are not interested in a lot of details at present. The promoter construct plus any inducer required (if the promoter is inducable) will be mixed together and the levels of fluorescence will be monitored over time.
We preferably want to use a constitutive promoter for Cell by Date. If all constitutive promoters are found working then we will need to chose one of them. The dicision can be made based on their levels of activity (the higher, the better) and their operating temperature range. This needs to be as close as possible to the CBD operating range of 4oC - 37oC
For Infector Detector, only the Lux promoter can be used so it is needed to work in vitro.
Phase 2 - Device construct
In phase 2, we will be carrying out experiments to actually test our two applications based on their specifications.
Cell by Date
In brief, we will be testing Cell by Date to see if it meets the specifications set during the design stage. This means that the device must be able to operate reliably for at least 24 hours and have a temperature operating range close to 4°C- 37oC. The device behaviour at step and impulse wise temperature changes must also be examined.
The first thing to be done before starting the expreriments is to incorporate DsRed-Express in the place of GFP for the promoter construct used in phase 1. This will produce the final device construct to be tested. To be able to correlate fluorescence measurements with the concentration of DsRed-Exp protein, we need a calibration curve. This can be obtained by measuring the fluorescence of varying purified DsRed-Exp concentrations.
Next we will carry out a simple test run of our device under room temperature. This is just to verify that our device actually works in vitro systems. The optimum temperature for the cell extract used is 24 oC- 37oC so room temperature will provide sufficient levels of expression. The maturation for DsRed-Exp is only 0.7 hours as opposed to 11 hours for normal Ds-Red so we expect to measure considerable fluorescence early in our experiments.
Following that our device works in vitro, we will carry out a set of experiments to determine the system behaviour at different temperatures. We will allocate the temperatures to be tested based on the specifications. To maintain constant temperatures across our systems from the start time of expression, we will use a series of water baths/incubators. Then for every half an hour, we will take each system and test its fluorescence levels. If the lab closes before the system comes to a halt, we will simply continue measurements the next day and extrapolate. From these experiments we aim to find out the operating range of our device as well as the rise time and expression life span of our system. Parallel to the experiments above, we will be testing the DsRed degredation rate at these various temperatures. This is done by keeping purified DsRed samples of similar concentrations along with our vitro systems in the water baths and incubators. So when we take our system out to measure, we will also be taking with us the purified DsRed samples. This will be an invaluable information when we do our modelling.
The final part of our CBD experiments will be to test the system responce when we have a temperature change while the system is in steady state (step change). Also the response to a sudden temperature surge (impulse) will be useful so as to know the limits of our system. The response to a gradual temperature increase will also be investigated.
When the vesicles to be used for the veso system have been successfully developed, we will carry out the same procedures as above to test CBD in veso.
Phase 3 -Validation device construct
The final phase will be based on the comparison between the modelling and our actual results. I.e. If they correlate or if they deviate and why ? Possible re-design of experiments will be cosidered.