Part:BBa_K2991016
pRbsD YFP reporter
Description:
This part allows you to study pRbsD expression with YFP.
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).
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.
See more results on our wiki page.
I - Testing of the functionality of our designed parts
After transforming K12 MG1655 E.coli with our pRIB-TFP 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 sugar. 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.
● pRIB-YFPE.coli :
Figure 4: YFP Fluorescence normalized with OD for E.coli K12 MG1655 transformed with pRIB in the presence (yellow) or absence (grey) of ribose at 0,2%. Experiments were conducted during 17 hours.
The activity of pRIB is relative to the expression of YFP fluorescence. In the condition “without sugar”, there is little increase in YFP. There is therefore no significant promoter activity in the absence of sugar. This validates our control. With 0.2% ribose, starting from the 4.5th hour, we observe a strong increase in YFP which is significantly different than the YFP observed in the condition without sugar. Therefore, we can confirm that ribose activates the expression of YFP by increasing the activity of pRIB. This verifies the proper functioning of our designed pRIB promoter.
Summary
pRIB is significantly activated in the presence of it associated sugar compared to the condition without sugar, verifying the proper functioning of this part.
II - The link between sugar concentration and promoter activity
For these tests we measured the fluorescence in K12 MG1655 E.coli transformed with our pRIB-YFP construct. The bacteria were cultivated in M9 medium with various different concentrations of the associated sugar. The fluorescence studied in the graphs below has been normalised with the Optical Density (OD).
● pRIB-YFP E.coli :
Figure 12: YFP Fluorescence normalized with OD for E.coli K12 MG1655 transformed with pRIB in selected concentrations of Ribose.
The activity of pRIB is relative to the expression of YFP fluorescence.
In the condition without sugar, we observe a very low, stable and significantly different YFP fluorescence compared to the YFP in the other conditions (YFP max is around 10 000 a.u compared to 40 000 and 70 000 for the other conditions).
In the conditions with sugar, we notice the same curve tendencies with an increase in YFP fluorescence around the third hour, however there is no significant difference of YFP levels between these different conditions.
We do notice one anomaly for the condition 0.01% ribose. At hour 12, the YFP fluorescence strongly increases (from around 40 000 au to 70 000 a.u.) during the last 4 hours of the culture.
We suspect either a measurement anomaly on these plate wells, an error during the preparation of the medium for this condition, or an unidentified phenomenon (this experience was repeated twice and the same curve was observed both
times).
The promoter pRIB was indeed activated in the presence of ribose during this experiment, however, does not seem sensitive to the change of concentration of this sugar. The intensity of the activity of pRIB in our construct does
not seem to depend on the concentration in ribose present in the medium.
Summary
In our simple insert constructs,pRIB, a high level of activation was quickly reached with a low concentration of their cognate sugars.
III - The specificity of each promoter to their associated sugar
To complete our experiments we tested the promoter activity of the simple insert constructs pRIB-YFP, in K12 MG1655 E.coli, in the presence of saturated concentration of different sugars. The primary purpose of these measurements is to confirm already published cross-activation results.
● pRIB-YFP E.coli :
Figure 18: YFP fluorescence normalized with the OD in E.coli K12 MG1655 bacteria transformed with pRIB-YFP construct in the presence of saturating amounts of different sugars.
The activity of pRIB is relative to the expression of YFP fluorescence.
In the condition without sugar we notice an increase in YFP over the first 5 hours which then stagnates after hour 5. This is similar to what was observed in Figures 7b and 8b where without sugar, there is a slight but sharp increase
of RFP fluorescence corresponding to pRIB activity over the first 5 hours before stabilization of the fluorescence. We can hypothesize that this increase could be due to internal ribose produced by the bacterie and that enters in the composition
of its DNA or RNA.
Only three sugars, arabinose, sorbitol and ribose were able to induce an YFP fluorescence higher than the condition without sugar. Maximum activation of pRIB is reached with its cognate sugar ribose. On the other hand, activation
of pRIB by lactose follow a very similar pattern as the negative control (in the absence of sugar). Hence, lactose does not seem to induce pRIB promoter activity. This last observation corroborates already published data by Guy Aidelberg et al (1).
Summary
pRIB is only activated by 3 sugars (arabinose, sorbitol and ribose) and is not activated by lactose. These confirm cross-activation data published previously by Guy Aidelberg et al (1).
We hypothesize
that internal bacterial ribose produced for DNA and RNA may explain the sudden and slight activation of pRIB promoter in the absence of any added sugar in the medium.
IV - Binary combinations of sugars with simple and double inserts
Here we tested the activities of single insers pRIB-YFP with binary combinations of sugars. The primary purpose of this experiment is to analyse the activity pRIB 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 constructions in this condition, and how a sugar, higher or lower in the hierarchy impacts the expression of the promoter.
● pRIB-YFP E.coli :
As a reminder, here E.coli K12 MG1655 was transformed with pRIB-YFP. We monitored pRIB activity by measuring YFP fluorescence in absence or presence of combinations of sugars. Results are provided below (Fig. 24).
Figure 24: YFP Fluorescence normalized with OD for E.coli K12 MG1655 transformed with pRIB-YFP in absence of any sugar (■), in the presence of 0.2% ribose combined with M9 rich medium (●), 0.2% lactose (○), 0.2% arabinose (△) and 0.2% sorbitol (+). Experiments were conducted during 17 hours.
Here, for all tested conditions, there is a sharp increase in YFP fluorescence during the first 2.5 hours. This is similar to our previous results as shown in Figures 7b, 8b and 18. As mentioned above, we hypothesize that this could result from endogenous
ribose produced by the bacteria for DNA and RNA that could activate pRIB promoter.
When 0.2% ribose was combined with M9 rich medium or 0.2% lactose, no additional increase of YFP fluorescence was observed when compared to the fluorescence in absence of sugar, indicating that these may strongly inhibit the action of
ribose on pRIB promoter. On the other hand, sorbitol and arabinose when combined with ribose did not inhibit pRIB promoter activity. Combined with arabinose, YFP fluorescence levels reached slightly higher levels than when combined with sorbitol.
We can conclude that in the simple construct, a sugar higher up in the hierarchy will inhibit promoters associated to sugars lower in the hierarchy. All sugars will induce an inhibition of the pRIB. This results were expected.
V - Duration of the promoter expression
As our tool is mainly based on controlling gene expression in time, it is important for us to study the activity of these promoters in time. This will allow us to better understand the behavior of our promoters, enabling us to take into account any time differences we might discover in the design of our tool.
● pRIB-YFP E.coli :
Figure 29: Rate of YFP Fluorescence/hours for E.coli K12 MG1655 transformed with pRIB-YFP simple insert in the absence or in the presence of 0,2% of Ribose
The activity of pRIB is relative to the expression of YFP fluorescence.
We do not observe an increase in the YFP production rate in the condition without sugar. It confirms our control. With 0,2% of Ribose, we can observe a linear increase of YFP production rate starting at the third hour. We have a peak in the YFP production rate at the 6th hour, and then the speed of YFP production declines until the 10,5th hour.
We can conclude that the activity of the promoter pRIB starts 3 hours after the presence of sugar in the medium and it is activated during 7,5 hours.
Summary
We can conclude that the activation and the activity of promoters vary in time, from one to the other. pRIB was activated around hour 3 and lasted around 7,5 hours.
This information would need to be taken into account to increase the precision of sequential gene expression in time of our tool.
(1)“Hierarchy of non-glucose sugars in Escherichia Coli.” Aidelberg Guy et al, BMC Systems Biology., 2014 , PMID: 25539838