IGEM:IMPERIAL/2007/new pages/DNA concentrations2
Testing DNA concentration of pTet-GFPmut3b In vitro
To determine if the optimum concentration of pTet-GFPmut3b in vitro.
Materials and Methods
Refer to protocols page.
Tested on (Insert link)
- Negative Control- Nuclease Free Water was added instead of DNA
- Temperature - 25°C
- Total Volume - 60µl
Figure 1.1 and figure 1.2 show that molecules of GFPmut3b increase with increasing DNA in our in vitro chassis. However, after 2µg the difference in molecules of GFP produced becomes very small and when the standard dievation is taken into account the differences looks like it is negligible.
The different DNA concentrations have a similar shape graph on figure 1.1. In addition the 2µg,4µg and 6µg show very similar response.
When the results for this experiment are compared to a similar experiment carried out for [[PUT LINK IN HERE TO DNA CONC pTet-LuxR-pLux-GFPmut3b] the difference in optimum DNA concentration becomes apparent. The pTet-LuxR-pLux-GFPmut3b showed an optimum at 4µg, above which the output of GFP molecules decreases. This is not the case for pTet-GFPmut3b which although does not see an increase above 4µg, it does not show the decrease.
The reason for this difference must be in the constructs used, pTet-LuxR-pLux-GFPmut3b is a large construct that has produces two proteins; pLux and GFPmut3b. In contrast the pTet-GFPmut3b is a smaller construct that only produces GFPmut3b. Promega quote the optimum for the commercial cell extract is 4µg, above which there is a decrease in protein synthesis because of premature termination of translation. One reason why the pTet-GFPmut3b does not show this decrease after 4µg is because there is only one protein being synthesized compared to the two of pTet-LuxR-pLux-GFPmut3b.
The optimum DNA concentration that was tested is between 2-6µg of DNA, however the difference between 2µg,4µg,6µg is negligible.