The optimum DNA concentration for [http://partsregistry.org/Part:BBa_T9002 pTet-LuxR-pLux-GFPmut3b] in our Commcercial S30 Cell extract is 4µg.
Aims
The aims of the testing were as follows:
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.
Both of these are important, first to try to optimise infector detector to reach the full potential of in vitro chassis and secondly to the specifications for the sensitivity to AHL.
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 Click here for results and protocol.
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 for 50nM AHL. 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 output of GFPmut3b synthesis:
Linear phase - The DNA Concentration is proportional to synthesis of GFP molecules
Saturation phase - The expressional machinary is saturated i.e. RNA polymerases and ribosomes, and so the synthesis is no longer affected by DNA concentration. The maximum synthesis of GFP is at 4µg.
Inhibition phase- Increasing the DNA concentration actually inhibits the rate of protein synthesis.
The fact that increasing DNA concentration above 4µg causes a decrease in rate of protein synthesis is very interesting. The reason for this is thought to be that increasing DNA concentration causes problems with premature translational termination.
For more information on the results please go the the results page
The 4µg was taken as the maximum and used for the rest of the testing.
AHL Testing
Fig.1.3: Molcules of GFPmut3b synthesised vs AHL concentrations. The Testing was carried out for the 4µg of DNA. Again the fluorescence reading was converted into GFPmut3b molecules via the calibration curve. Click for full results and protocols can be found on the links results and protocol pages.
Fig.1.4:Molecules of GFPmut3b synthesised for each DNA Concentration in vitro, after 360 minutes. The data is from the fluorescence measurement at 360minutes which was converted to GFPmut3b molecules and plotted against [AHL]. The scale on the X axis is a logarithmic scale.
Figure 1.3 shows 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 proved degradation is negligible. Interestingly this time at which synthesis stops 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.
Figure 1.4 shows us the following:
The shape of the Transfer function shows a linear range of response between 5nM and 100nM AHL. This defines the thresholds of response.
The lower threshold of response is the AHL concentration that the construct will respond
The upper threshold of response is the value of AHL the system is saturated and increasing AHL will not increase the rate of GFP synthesis.
For more detailed analysis please see the Results page
