Part:BBa_K3463004:Experience
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Characterisation BBa K3463004
In order to compare the strength of three promoters, we have monitored four DH5 alpha cultures:
DH5a-pUCBB-J23100- eGFP BBa_K3463006
/ DH5a-pUCBB-J23102-eGFP BBa_K3463004 / DH5a-pUCBB-J23106- eGFP BBa_K3463005 / DH5a (Control -)
These 4 cultures were inoculated in triplicata (OD600nm=0.1) in a semi-transparent 96-wells plate and incubated at 37°C in Varioskan LUX six hours long. This experiment was repeated three times and data were collected and analysed together to build the following graphs.
Every ten minutes, both OD600nm (Figure 1) and eGFP fluorescence (Figure 2) were measured in each well and triplicata's mean were plotted.
Averages: DH5 wt=J23100=J23102=J23106
Growth curves are presented Figure 1. We can see that all these curves have the same pattern of evolution over time. Therefore, when compared to the growth curve of the control culture, we can conclude that the strength of a promoter has no effect on the growth of the bacterial culture (p-value=0.896).
The figure 2 shows the fluorescence intensity over time. For each curve, the first measurement point (T0) was set at 0 and subtracted to the following ones. Firstly, no fluorescence was detected in the DH5 alpha wt control culture. In contrast, in all the cultures expressing eGFP under the control of a specific constitutive promoter, we detected an significant increasing amount of fluorescence over time (p-value<2.10-16). The total amount of eGFP produced increased according to the promoter tested with 15.2, 9.7 and 2.5 RFU for J23100, J23102 and J23106 respectively. Significantly, the promoter J23100 seems to be the best one to produce eGFP in large quantities. Thus, it will be used to produce RhlR BBa_K3463007 .
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