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In vivo Testing of pTet-LuxR-pLux-GFPmut3b Construct

Aims

To determine if the of pTet-LuxR-pLux-GFPmut3b construct works in vivo.

Materials and Methods

See Protocols page

Results

Fig.1.1:pTet-LuxR-pLux-GFPmut3b in vivo - The construct pTet-LuxR-pLux-GFPmut3b was tested in vivo to determine if the construct works. E.coli BL21 was grown and induced with varying levels of AHL. Two samples were taken every hour from batch cultures, one of these samples was lysed by freeze-thaw and the other whole cells were measured.

Fig.1.2:Growth of in vivo - Shows the average growth of the cultures used in this experiment.

Controls:

• Negative Control- E.coli BL21 containing an empty vector from the registry

Constants:

• Temperature - Cells grown at 37°C
• Volume of Cells sampled - 500µl
• Volume Measured - 60ul of PBS buffer

Raw Data

• Raw Data excel file

Discussion

Figure 1.1 shows that the construct pTet-LuxR-pLux-GFPmut3b works in vivo. The results show that with increasing [AHL] the fluorescence increases, therefore the GFPmut3b produced is increasing with increasing [AHL]. The negative control increases slightly, however remains very low in comparison to the samples.

The difference between the lysed and whole cells does not have a clear pattern, for some samples the lysed cells are higher and in other samples it is lower. This variability is likely to be due to the experimental method, because the lysed and the whole cells where different samples and so error could have been introduced.

The O.D.₆₀₀ shows that the growth of the cells containing the empty vector and pTet-LuxR-pLux-GFPmut3b were in the exponential growth phase that appeared to be moving into the steady state phase at the end of the experiment. There is no clear difference between the empty vector or the vector containing pTet-LuxR-pLux-GFPmut3b, this means that the pTet-LuxR-pLux-GFPmut3b appears to have very little metabolic burden on the cells.

Conclusion

The pTet-LuxR-pLux-GFPmut3b is working in vivo and GFPmut3b produced increases with increasing [AHL].