Difference between revisions of "Part:BBa K2448032"

Latest revision as of 16:18, 25 October 2020

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 reactions involved in the working of the biosensor constructs are

Parameters

Differential Equations

Here,

[R] is the FruR concentration; [M] is the mCherry concentration; [C1] is the concentration of the [F1P-FruR] complex; [C2] is the concentration of the [pFruB-FruR] complex (in the binding domain); and [CB]=CN/V is the approximate concentration of binding domains per cell where CN is the plasmid copy number and V is the cell volume.

Note: We were aware of probability-based techniques to perform parameter estimations (such as Bayesian parameter estimation), but as there were a large number of undetermined parameters in our model, it would not make any stastical sense to estimate such a large number. The parameter space would be too large for us to derive any significant or meaningful results. Therefore, we chose to limit ourselves to a theroretical description which we hope teams in the future can use and improve upon.

Sequence and Features