User:James Chappell/Growth phase

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Fig.1.1:Growth of E.colicontaining the J45995 device (stationary phase sensitive) and J45996 (exponential phase sensitive)at 37oC - The growth is measured at OD600 for 24 hours
Fig.1.1:Growth of E.colicontaining the J45995 device (stationary phase sensitive) and J45996 (exponential phase sensitive)at 37oC - The growth is measured at OD600 for 24 hours
Fig.1.2:Normalised GFP vs time (6 hours) for E.coli containing J45995 device (stationary phase sensitive) and J45996 (exponential phase sensitive), see below for explanation of Normalised [GFP]
Fig.1.2:Normalised GFP vs time (6 hours) for E.coli containing J45995 device (stationary phase sensitive) and J45996 (exponential phase sensitive), see below for explanation of Normalised [GFP]

Fig.1.2:Normalised GFP vs time (24 hours) for E.coli containing J45995 device (stationary phase sensitive)and J45996 (exponential phase sensitive), see below for explanation of Normalised [GFP]
Fig.1.2:Normalised GFP vs time (24 hours) for E.coli containing J45995 device (stationary phase sensitive)and J45996 (exponential phase sensitive), see below for explanation of Normalised [GFP]
Figure 1.1 it can be seen that the growth rate of the E.coli containing the J45996 is higher than that of E.coli J45995. The reason for this seems unclear and if anything unexpected. The J45996 involves greatest burden of synthesis in the exponential phase and also has the added burden of expression associated with the inverter. One cause for this difference seen could be due to different cell densities in the overnight cultures added to fresh media at time 0.

Figure 1.2 and 1.3 shows the normalised [GFP] vs time. Normalised [GFP] was calculated by normalising by the OD600 and converting fluorescence into GFP using a calibration curve. As can be seen both cultures start with the a initial level of [GFP], this is due to synthesis in the overnight culture which is still present at time 0. From the GFP changes little for the first hour until cells begin to divide and a degradation term is introduced into our system (see simple model below). The degradation until synthesis begins for both constructs when cells enter early and late exponential phase. Overnight we see that the exponential phase promoter (J45996) maintains the steady-state seen at 6 hours overnight. The stationary phase promoter (J45995)increases in [GFP] overnight as the cell population enters the stationary phase and is most likely in a steady-state.
There are several key features that can be drawn out of this data, first the different devices have two clear steady-states, with stationary phase promoter appearing to have a higher steady-state. This is due to the fact that when the promoter is activated there is effectively no degradation term (from cell division), in comparison in the exponential phase there is a degradation term of approximately 40 minutes (every cell division).
There was no negative control for this experiment so it is difficult to know if the apparent steady-state of J45996 is actually a steady-state or just background fluorescence. Clearly if this was to be repeated inclusion of a negative control would be crucial.

There is a lot of variation within the data collected for both figure 1.1 and 1.2. The reason is firstly because of the intrinsic variation of expression in E.coli. In addition this data was a combined effort of Imperial College London Synthetic Biology Course and was collected by a group of around 30 engineers and biologist. This combined data introduces experimental variation that will have contributed to the variation seen.




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