Part:BBa_K2991001

pSrlA regulated promoter Sorbitol

Description:

Promoter srlA with its regulatory sequence (as detailed on the Ecocyc webSite) to induce transcription in presence of sorbitol. It includes the normal CRP fixation site.

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
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 our designed parts

After transforming K12 MG1655 E.coli with our 6 different constructs, 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.

● pSRL-RFP E.coli :

Figure 3: RFP Fluorescence normalized with OD for E.coli K12 MG1655 transformed with pSRL in the presence (red) or absence (grey) of sorbitol at 0,2%. Experiments were conducted during 17 hours.

The activity of pSRL is relative to the expression of RFP fluorescence. We do not observe a significant difference of RFP fluorescence between both conditions, with or without sorbitol. Either (i) there is a RFP fluorescence in both conditions meaning that the promoter pSRL is active even in the absence of sugar, or (ii) we are in presence of an experimental error.

Summary

Unfortunately, we were not able to have pSRL construct to work properly.

II - The link between sugar concentration and promoter activity

For these tests we measured the fluorescence in K12 MG1655 E.coli transformed with our pSRL-RFP. 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).

● pSRL-RFP E.coli :

Figure 11: RFP Fluorescence normalized with OD for E.coli K12 MG1655 transformed with pSRL in selected concentrations of Sorbitol

The activity of pSRL is relative to the expression of RFP fluorescence. This figure shows no significant difference of RFP expression between all the different conditions This is coherent with our previous observations whereby we observed that pSRL activity was equivalent in absence and in presence of 0.2% sorbitol (see figure 3). The pSRL does not seem to be affected by the sugar in the medium, therefore we assume a dysfunctionality of this promoter in our constructs

Summary

Unfortunately, we were not able to have pSRL construct to work properly.

III- Binary combinations of sugars with simple and double inserts

Here we tested the activities of single inserts with binary combinations of sugars. The primary purpose of this experiment is to analyse the activity of pSRL 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 constructions in this condition, and how a sugar, higher or lower in the hierarchy impacts the expression of the different promoters.

Summary

The pSRL results are not exploitable.