To test and obtain the optimal DNA concentration for construct 1 in vitro
To characterise the output of GFPmut3b for a range of AHL inputs. From this obtain the AHL sensitivity of our system.
In addition the fluorescence measurements were converted to number of GFPmut3b molecules synthesised using a calibration curve constructed using purified GFPmut3b.
Results
DNA Concentrations
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.
The Results above show that the optimum DNA concentration for in vitro is 4µg. From figure 1.1. and 1.2 it can be seen that as DNA concentration increases above 4µg the GFPmut3b molecules synthesised decrease. Interestingly for figure 1.2 the graph can be split into several regions of how the DNA concentration changes the GFPmut3b synthesis. Between
Testing AHL Range
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
