Part:BBa_K2448032
Fructose Biosensor pFruB-FruR from Escherichia coli
This part is a Fructose Biosensor based on the FruR transcription factor from Escherichia coli (BBa_K2448009) and its associated promoter pFruB (BBa_K2448017).
Usage and Biology
Biosensors rely on a basic theoretical principle: a certain concentration of a molecule of interest induces the proportional production of an easily detectable output, like fluorescence. Transcription-factor based biosensors allow quick and cheap detection or quantification of various chemical compounds.
Features
This biosensor was built using the Universal Biosensing Chassis (BBa_K2448023, BBa_K2448024) which is a composite part that provides an answer to the lack of rapid and reliable building methods for transcription-factor based biosensors.
It is based on the FruR transcription factor from Escherichia coli (BBa_K2448009) and its associated promoter pFruB (BBa_K2448017).
FruR from Escherichia coli (BBa_K2448009) is a LacI family transcription factor with affinity for D-Fructose. FruR is capable of binding a consensus sequence in the promoter region and preventing transcription of the regulated promoters in the absence of D-Fructose, in a similar manner to the way LacI does in the absence of allolactose (or the synthetic IPTG).
In this biosensor, we used this Helix-Turn-Helix transcription factor together with the Escherichia coli pFruB (BBa_K2448017) which is the promoter region (0.25 kb upstream) of the FruB gene of Escherichia coli K-12 (ECK2162). pFruB is repressed by the FruR transcription factor (BBa_K2448009) which is inhibited in the presence of D-Fructose. This promoter regulates the expression of mCherry in our biosensor.
The results presented hereafter show that this duo FruR - pFruB behaved as predicted under and without induction.
Principle
When pTacI is induced by IPTG, it drives the transcription of the FruR gene coding for the FruR protein which is a transcription factor able to bind D-Fructose. If D-Fructose is present in the cell, the transcription factor will bind preferentially to it and thus be inactivated. The repression of the related promoter pFruB will be released, enabling the transcription of a fluorescent protein, mCherry. If D-Fructose isn’t present in the cell, FruR will bind to pFruB, preventing any transcription of mCherry.
Characterization
The detailed protocol is presented in the Experience page.
Optimal measurement time
The characterization of the biosensors allowed us to determine many important parameters. For instance, running the experiment for a long period (18 hours) got us an estimation of the optimal measurement time.
To estimate this duration, we looked at the raw data and observed that it took around 9 hours to get an observable signal for the lowest concentration of inducer. It means that sensitivity threshold and consequently maximum accuracy is reached 9 hours after induction.
Since we wanted to detect and measure D-Fructose concentration between 10 mM and 300 mM, we needed a biosensor able to get maximum accuracy for this range of concentration in a minimal time. Taking into account the raw data, we can estimate that for this biosensor if D-Fructose concentration is above 10 mM, a 5 hour incubation after induction would give relevant results.
Basal expression
The biosensor show a basal expression of 4000 arbitrary units of fluorescence at 18 hour post-induction. This basal activity even without fructose in the media is due to an imbalance between the amount of FruR transcription factor available and the pFruB promoter strength. Even when FruR is produced, the transcription factor can’t totally prevent the transcription from happening.
Dynamic range
Determining the dynamic range of our biosensor will give us an estimate of its sensitivity, its maximum and its potential use. We can observe in figure 2 a perfect foldchange and perfect linearity in range of concentrations from 10 mM to 300 mM.
This gives us two important pieces of information:
- First, the results show that FruR interact with fructose The same observation applies for the pFruB promoter which seems tightly regulated by FruR under fructose induction.
- Second, the dynamic range of this biosensor appears to go from 10 mM to 300 mM fructose. This means that we will be able to use it in real applications for our bioscreening protocol to assess the production of fructose that could range from 1 mM to maximum 300 mM.
Figure 2. In vivo characterization of this Fructose Biosensor in E. coli TOP10. The graph shows the mCherry measured florescence over fructose concentration in the media. Each data point is the mean of two technical duplicates and of three biological triplicates. Error bars represent standard deviations.
BITSPilani-Goa_India Contribution
Mathematical Model
The assumptions we have made for the model are listed below:
1. As cells would be IPTG-induced before being added to the inoculant, we have assumed that the total number of FruR molecules remains constant during the course of the action of the cells.
2. Phosphorylation and dephosphorylation of fructose is not considered to simplify the model. We assume that all the fructose that is taken up by the cell is immediately phosphorylated. Hence, the amount of fructose is roughly equal to the amount of fructose-1-phosphate. Subsequently, the metabolic pathway from F1P leading to FBP, F6P, G6P is also ignored (Chavarría et al., 2014).
3. The FruR-F1P complex is higly stable. This ensures that the FruR in the complex is not recycled back such that it can preferentially bind to pFruB.
4. The binding rate for F1P and FruR is unaffected by pfruB.
5. FruR only binds to pfruB when it is bound to the DNA sequence.
6. F1P binds to FruR and helps it unbind from pFruB.
7. There is a constant external source of FruR and pFruB which maintains their concentrations at a steady, constant level throughout the timescale of the model.
Parameters
Differential Equations
Here, [P] is the pFruB concentration; [R] is the FruR concentration; [M] is the mCherry concentration; [A] is the anti-invertase concentration; [C1] is the concentration of the [F1P−FruR] complex; [C2] is the concentration of the [pFruB−FruR] complex (in the binding domain); and [Pb] is the concentration of pFruB bound to the binding domain (Bisswanger, H., 2008) [Rb] is the concentration of the domain-bound pFruB and FruR complex
Molecular Modelling
We docked FruR and Fructose-1-Phosphate using AutoDock.
We also docked the modelled FruR-pFruB complex with Fructose-1 phosphate using AutoDock. We observed that there were no significant interactions taking place which meant that there is a conformational change induced by pFruB on FruR. We can see that the major fructose binding pocket undergoes a conformational change (shown by the dis-alignment of the initial blue beta sheet and the new red beta sheet) leading to a changed beta sheet structure.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1262
Illegal XhoI site found at 62 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2129
Illegal AgeI site found at 2241 - 1000COMPATIBLE WITH RFC[1000]
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