Part:BBa_K2991006

Hierarchized double promoter ( pLacI-pSrlA ) with GFP/RFP

Description:

This composite part allows you to simultaneously visualize the expression of pLacI and pSrlA. This part is designed to study the influence of a complex medium containing multiple sugar. You will indeed be able to measure, with the two different fluorescents proteins, the hierarchy of expression for its associated promoter.

What you can do with it:

In E.coli sugars are ranked depending on their capacity to increase growth rate. Promoters associated with each sugar operon will follow this hierarchy. Each part of the collection is made to exploit this Hierarchy (check our wiki, https://2019.igem.org/Team:Nantes).
See more results on our wiki page.
You will learn more about it in this study ‘’G. Aidelberg, B. D. Towbin, D. Rothschild, E. Dekel, A. Bren, and U. Alon, “Hierarchy of non-glucose sugars in Escherichia coli,” BMC Syst. Biol., vol. 8, p. 133, 2014.’’

Compatibility:

Organism: We only tested this part in E.coli K12 MG1655.
Backbone: We used the IDT plasmid with ampicillin resistance.

I - Testing of the functionality of pLAC-GFP/pSRL-RFP

After transforming K12 MG1655 E.coli with our pLAC-GFP/pSRL-RFP construct, we measured by spectrofluorometry the fluorescence of the transformed bacteria cultivated in M9 medium in the presence of saturating concentration (0.2%) of the associated sugars (Lactose and Sorbitol)The fluorescence studied in the cases below has been normalized with the Optical Density (OD).

Our spectrofluorometric measurements were carried out on the transformed bacteria cultivated in a M9 medium in 2 different conditions : with 0.2% one of the specific sugars, and without sugar.

● pLAC-GFP/pSRL-RFP E.coli:

The activity of pLAC is relative to the expression of GFP fluorescence, and the activity of pSRL to RFP fluorescence.

Measurements done in the presence of 0.2% lactose and without sugar:

Figure 5: GFP(a) and RFP(b) fluorescence in K12 bacteria with the double insert (pLAC-GFP + pSRL-RFP), in the absence (grey) or presence of 0.2% lactose (green for GFP or red for RFP). Experiments were conducted during 17 hours

In figure 5a, as expected, we observe a significant difference between GFP levels in the presence of lactose and without the sugar. The pLAC in our double insert construction is not activated in the absence of sugar (as previously shown for the single insert construction, Figure 1).
Likewise, in Figure 5b, in the absence or presence of lactose there is no difference in the emission due to RFP. Lactose did not induce an increase in the pSRL promoter activity.
Therefore, pLAC is significantly activated in the presence of lactose, however the presence of lactose does not affect the activity of pSRL

Measurements done in the presence of 0.2% sorbitol and without sugar:

Figure 6:GFP(a) and RFP(b) fluorescence in K12 bacteria with the double insert (pLAC-GFP + pSRL-RFP), in the absence (grey) or presence of 0.2% sorbitol (green for GFP or red for RFP). Experiments were conducted during 17 hours

In Figure 6a we notice an increase in GFP fluorescence with 0.2% sorbitol and no increase of the GFP fluorescence in the absence of the sugar. This means that sorbitol is able to activate pLac promoter and this corroborates the data published in the paper of Dr. Guy Aidelberg et al (1).

In figure 6b, we notice that sorbitol even in saturating concentrations (0.2%), do not activate pSrl promoter and hence RFP fluorescence in the context of a double insert with the pLac promoter. There could be two explanations for this observation. First, it could be that possible our pSrl promoter is not functioning. This would explain our data in figure 6b and 3. Indeed, the promoter in the single insert construction (see above) is not activated by sorbitol

The fact that sorbitol strongly activates the pLac promoter as shown by GFP fluorescence, could also mean that sorbitol would preferentially activates pLac promoter in place of pSrl promoter. This result has never been shown before because no binary combination of both of these promoters were tested before. Our data could suggest that there is indeed a hierarchy in the activation of promoters. A certain amount of Sorbitol will activate pLac if the latter is present. We cannot so far say if both promoters could be activated and to which level one compared to the other because our pSrl insert does not seem to function

Summary

I
In the case of this double insert construct, we observed an activation of pLAC, in the presence of it respective sugars.
Thus confirming the existence of cross-activation and a sugar hierarchy in E.coli.

II - The link between sugar concentration and promoter activity

For these tests we measured the fluorescence in K12 MG1655 E.coli transformed with our pLAC-GFP/pSRL-RFP. The bacteria were cultivated in M9 medium with various different concentrations of the associated sugars (Lactose and Ribose). The fluorescence studied in the graphs below has been normalised with the Optical Density (OD).

● pLAC-GFP/pSRL-RFP E.coli :

The activity of pLAC is relative to the expression of GFP fluorescence, and the activity of pSRL to RFP fluorescence.

Measurements done in the presence of different concentrations of lactose and without sugar:

Figure 13:GFP(a) and RFP(b) normalized with OD for E.coli K12 MG1655 transformed with pLAC/pSRL in selected concentrations of Lactose

Without sugar, the GFP fluorescence does not significantly change (very slight increase from 400 au to 500 a.u.), and remains significantly inferior to the GFP fluorescence in presence of lactose.
The GFP signal is lower with 0.01% lactose in comparison to the signals observed with higher concentrations of the sugar (figure 13a). There is no significant difference in GFP fluorescence levels between experiments with lactose concentrations at 0.02%, 0.05% and 0.1%. Maximal activation of pLAC promoter seems to be reached as from 0.02% lactose.
The RFP fluorescence does not seem to have any increase over time, or to have any significant difference between the different conditions. Therefore, there is no significant pSRL activity due to the presence of lactose. It might be because the pSRL promoter is not functional as it was seen in Figures 3, 5b and 6b. So we can conclude that we were not successful to have a function pSRL promoter.

Figure 14:(a)GFP and (b)RFP Fluorescence normalized with OD for E.coli K12 MG1655 transformed with pLAC/pSRL in selected concentrations of Sorbitol

In Figure 14a we observe that the GFP without sugar and with 0.01% sorbitol are not significantly different. 0.01% sorbitol is not enough to activate pLAC promoter. Significant activation of this promoter is only observed after 6 hours for concentration of sorbitol of 0.02% or above and would be concentration dependent. Though maximal activation is reached with 0.05% sorbitol
In figure 14b we observe no significant difference between any of the sorbitol concentrations. As mentioned above for Figure 13b, our results seem to indicate that pSRL promoter is unfortunately not functional

Summary

In the case of this double insert construct, we observed an activation of pLAC, in the presence of it respective sugar.