To test and characterise the key characteristics of our system such as the sensitivity of the system to AHL. To do this, we induce the system with known concentrations of AHL input and measure the fluorescence output. Then using a calibration curve the fluorescence was converted into the number of GFPmut3b molecules synthesised, click on the following link for an explaination about how to use calibration curve use the calibration curve].
Fig.1.1:Molecules of GFPmut3b synthesised over time, for each DNA Concentration in vitro - The fluorescence was measured over time for each experiment and converted into molecules of GFPmut3b in vitro using our calibration curve.
Fig.1.2:Molecules of GFPmut3b synthesised for each DNA Concentration in vitro, after 360 minutes
Results
Click for full results and protocols can be found on the links results and protocol pages.
The results show us the following:
The output of GFPmut3b increases with input of AHL
The system is sensitive to a range of 5-1000nM AHL
The GFPmut3b molecules synthesis stops at ~300minutes. This could be due to steady state or due to no synthesis of GFPmut3b. It is known not to be steady state because the degradation experiment(link) proved degradation is negligible. Interestingly this time is independent of the GFPmut3b molecules produced, showing that the LuxR under the control of pTet is the major source of energy consumption. This highlights the advantages of using the construct 2 [http://partsregistry.org/Part:BBa_J37032 pLux-GFPmut3b] that does not have the energetic burden of producing LuxR
