Part:BBa_K2991007
Hierarchized double promoter ( pAraB-pRbsD ) with CFP/RFP
Description:
This composite part allows you to simultaneously visualize the expression of pAraB and pRbsD. 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 pARA-CFP/pRIB-RFP
After transforming K12 MG1655 E.coli with our pARA-CFP/pRIB-RFPconstruct, 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 (Arabinose and Ribose). 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% of the specific sugar, and without sugar.
● pARA-CFP/pRIB-RFP E.coli :
The activity of pARA is relative to the expression of CFP fluorescence, and the activity of pRIB to RFP fluorescence.
Measurements done in the presence of 0.2% arabinose and without sugar:
Figure 7: CFP(a) and RFP(b) fluorescence in K12 bacteria with the double insert (pARA-CFP + pRIB-RFP), in the absence (grey) or presence of 0.2% arabinose (cyan for CFP or red for RFP). Experiments were conducted during 17 hours.
We observe in Figure 7a an increase of CFP fluorescence in the presence of 0.2% arabinose after a latency of about 4.5h and no increase of the same fluorescence in the absence of the sugar. This validates our control (without sugar) but also indicates that the pARA promoter in the context of the double insert is indeed activated by arabinose.
Figure 7b shows that for the first 8 hours, we do not have a significant difference in the fluorescence of RFP between the two conditions tested, i.e with or without 0.2% arabinose. After 8h, there is an increase in RFP fluorescence relative to that observed
in the absence of the sugar, indicating an increase in the pRIB promoter activity induced by the sugar. As detailed in the Dr. Guy Aidelberg et al (1) paper, arabinose can indeed very slightly activate pRIB promoter. This hence corroborates
their observations which was done with a single pRIB insert. It is noteworthy that in our case we have performed the measurements every 30 min during 17h while Aidelberg et al established activation level of pRIB by arabinose on a
single data point (at mid-exponential growth). Interestingly, the delay for the activation of pRIB by arabinose is 8h, twice the time needed for it’s activation with ribose only (see figure 4). This is an original result. Surprisingly,
we note a sharp increase in RFP fluorescence in the microplate wells with the double insert (pARA/pRIB) very early during bacterial growth from t=0h to t=2h. This increase may be artifactual due to some experimental error
Measurements done in the presence of 0.2% ribose and without sugar:
Figure 8: CFP(a) and RFP(b) fluorescence in K12 bacteria with the double insert (pARA-CFP + pRIB-RFP), in the absence (grey) or presence of 0.2% ribose (cyan for CFP or red for RFP). Experiments were conducted during 17 hours
We observe a significant linear increase of CFP in the presence of ribose when compared to the experiment without sugar, validating our control.
In the presence of ribose (Figure 8a), CFP fluorescence increases linearly throughout the experiment and has a maximum level at around 17 000 a.u obtained at hour 17. This means that ribose is able to activate the pARA promoter. This corroborates with the experimental data published by Guy Aidelberg et al (1).
When pRIB activity is monitored in the presence or absence of 0.2% ribose in the context of a double insert with pARA, there is a difference that is observed between the RFP fluorescence as from 4.5h. This confirms that pRIB promoter is indeed activated by ribose. Interestingly seems to be an immediate activation of pARA by ribose while there is a delay in the activation of pRIB by the same and similar to what was observed in figure 7b (activation of pRIB with arabinose that exhibited a delay of 4.5h).
Summary
In the case of this double insert construct, we observed an activation of pARA and pRIB in the presence of their respective sugars.
They are both activated but to a lesser degree in the presence of the other sugar, 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 pARA-CFP/pRIB/RFP. The bacteria were cultivated in M9 medium with various different concentrations of the associated sugars (Arabinose and Ribose). The fluorescence studied in the graphs below has been normalised with the Optical Density (OD).
● pARA-CFP/pRIB-RFP E.coli :
The activity of pARA is relative to the expression of CFP fluorescence, and the activity of pRIB to RFP fluorescence.
Measurements done in the presence of different concentrations of arabinose and without sugar:
Figure 15: CFP (a) and RFP (b) Fluorescence normalized with OD for K12 MG1655 transformed with pARA/pRIB in selected concentrations of Arabinose
We observe in Figure 7a an increase if CFP fluorescence in the presence of 0.2% arabinose after a latency of about 4.5h and no increase of the same fluorescence in the absence of the sugar. This validates our control (without sugar) but also indicates that the pARA promoter in the context of the double insert is indeed activated by arabinose.
In the conditions without sugar for both figures, we observe a stable fluorescence. Both promoters do not seem to be significantly activated in the absence of arabinose.
In both graphs, the fluorescence in the presence of arabinose become significantly different from the fluorescence in the absence of sugar from around hours 7 and 8 of the culture.
Our data do not show a concentration dependant variation of GFP or RFP fluorescence. Both pARA and pRIB promoters are very active at low concentrations of arabinose. This is an original result
Measurements done in the presence of different concentrations of ribose and without sugar:
Figure 16: CFP (a) and RFP (b) Fluorescence normalized with OD for E.coli K12 MG1655 transformed with pARA/pRIB in selected concentrations of Ribose
We observe a significant linear increase of CFP in the presence of ribose when compared to the experiment without sugar, validating our control.
In figure 16a, CFP fluorescence seems not to vary significantly between any of the conditions. There does not seem to be any particular activity of the pARA promoter in the presence of ribose in the medium.
In figure 16b, there is a non significant variation in RFP fluorescence in the absence of ribose. In presence of the sugar, the RFP fluorescence is increasing significantly after 6.5-7h. The increase is concentration dependant.
This experimental setup where the two promoters were combined and tested against ribose is original. Here the highest concentration of ribose used was 0.1%. It seems to be insufficient to activate the pARA promoter while it
was able to do it at a concentration of 0.2%. We can hypothesize that in the context of this double insert, ribose preferentially activates its own promoter pRIB and not pARA, when it is at a concentration of 0.1% or below. At
higher concentrations (like at 0.2%), the pRIB promoter is saturated and some ribose is available for activating pARA (Figure 8).
Summary
We observed an activation of pARA and pRIB in the presence of their respective sugars.
They are both activated but to a lesser degree in the presence of the other sugar, thus confirming the existence of cross-activation and a sugar hierarchy in E.coli.
III-Binary combinations of sugars
Here we tested the activity of our pARA/CFP-pRIB/RFP construct with binary combinations of sugars. The primary purpose of this experiment is to analyse the activity of each promoter in the presence of 2 sugars at a time: it’s own specific sugar and another. We will see if the sugar-hierarchy is still present in our construction in these conditions, and how a sugar,higher or lower in the hierarchy impacts the expression of the different promoters.
● pARA-CFP/pRIB-RFP E.coli :
As a reminder, here E.coli K12 MG1655 was transformed with a double insert containing pARA-CFP/pRIB-RFP. We monitored both pARA and pRIB activities by measuring CFP and RFP fluorescence respectively in absence or presence of combinations of sugars. Results are provided below (Fig. 25).
Figure 25: CFP Fluorescence normalized with OD for K12 MG1655 transformed with pARA-CFP/pRIB-RFP double insert in absence of any sugar (■), in the presence of 0.2% arabinose + 0.2% ribose (●), 0.2% arabinose + 0.2% lactose (○), 0.2% arabinose + 0.2% sorbitol (△), 0.2% ribose + 0.2% lactose (+) and 0.2% ribose + 0.2% sorbitol (◇). Experiments were conducted during 17 hours.
Results in figure 25 show that in absence of sugar, there is no significant change in CFP fluorescence. This validates the negative control for CFP fluorescence.
In presence of 0.2% arabinose combined with 0.2% lactose or 0.2% ribose, CFP fluorescence that is supposed to monitor pARA activity showed a steady increase as from 6 hours of culture. This suggests that neither lactose nor ribose inhibits
pARA promoter activity.
Similarly, when 0.2% arabinose was combined with 0.2% sorbitol, a steady increase in pARA activity as from the 7.5th hour was also observed but with much lower levels of CFP fluorescence reached when compared to the combination with lactose
or ribose.
The combination of ribose with lactose also induced a steady increase in CFP fluorescence as from 7.5 hours that reaches a level that is roughly 50% to that reached when arabinose was combined with lactose or ribose. Similarly, when 0.2% ribose was combined with 0.2% sorbitol, a steady increase in pARA activity as from the 7.5th hour was also observed but with much lower levels of CFP fluorescence as for arabinose + sorbitol.
Figure 26: RFP Fluorescence normalized with OD for E.coli K12 MG1655 transformed with pARA-CFP/pRIB-RFP double insert in absence of any sugar (■), in the presence of 0.2% arabinose + 0.2% ribose (●), 0.2% arabinose + 0.2% lactose (○), 0.2% arabinose + 0.2% sorbitol (△), 0.2% ribose + 0.2% lactose (+) and 0.2% ribose + 0.2% sorbitol lactose (◇). Experiments were conducted during 17 hours.
We also monitored the RFP fluorescence when the double insert construct was tested with combinations of sugars (Figure 26).
The results show that pRIB activity measured as RFP fluorescence was not significantly changed in all tested conditions. We can hypothesized that we did not measured any pRIB activity probably due to the fact that the sugars would either
have preferentially promoted pARA activity (e.g ribose or lactose) or inhibited pRIB activity.
We notice that every combination of sugar in a lower hierarchy than the arabinose produced a CFP fluorescence which results of the induction a pARA activity. It does not modified the activation of pARA. The combination of 0,2% of lactose and 0,2% of Arabinose shows an activation of pARA. It means that the Lactose did not inhibit the pARA, as we expected. It can be a cross-activation because theses sugars are close in the hierarchy. When we study the RFP fluorescence (showing the activity of pRIB), we can’t observe any significant results because every sugar are higher than Ribose in the hierarchy. All sugars will induce an inhibition of the pRIB. This results were expected.
(1)“Hierarchy of non-glucose sugars in Escherichia Coli.” Aidelberg Guy et al, BMC Systems Biology., 2014 , PMID: 25539838