Part:BBa_K2448025
Psicose Biosensor pPsiA-PsiR from Agrobacterium tumefaciens
This part is a Psicose Biosensors based on the PsiR transcription factor from Agrobacterium tumefaciens (BBa_K2448006) and its associated promoter pPsiA (BBa_K2448010).
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.
Psicose biosensors were the lynchpin of our iGEM project. In order to improve the enzymatic production of psicose, we needed an efficient method to screen large banks of mutants. To address this problem, we designed a collection of specific biosensors able to detect psicose concentration in vivo (BBa_K2448025, BBa_K2448026, BBa_K2448027, BBa_K2448028, BBa_K2448029, BBa_K2448030 an BBa_K2448031). This allowed us to correlate the fluorescence with the mutant enzyme activity. Combining this biosensor with a plate reader, we were able to screen a library of mutants and identify the cells carrying an improved version of our enzyme, the D-Psicose 3-epimerase (Dpe) from Clostridium cellulolyticum (BBa_K2448021. For more details see BBa_K2448035.
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.
This part is a Psicose Biosensors based on the PsiR transcription factor from Agrobacterium tumefaciens (BBa_K2448006) and its associated promoter pPsiA (BBa_K2448010).
PsiR from Agrobacterium tumefaciens (BBa_K2448006) is a predicted LacI family transcription factor with high affinity for D-Psicose. This implies that PsiR is potentially capable of binding a consensus sequence in the promoter region and preventing transcription of the regulated promoters in the absence of D-Psicose, 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 Agrobacterium tumefaciens pPsiA promoter (BBa_K2448010) which is the promoter region (0.4 kb upstream) of the PsiA gene of Agrobacterium tumefaciens str. C58 (Atu4744). pPsiA is predicted to be repressed by the PsiR transcription factor (BBa_K2448006)which is inhibited in the presence of D-Psicose. This promoter regulates the expression of mCherry in our biosensor.
The results presented hereafter show that this duo PsiR - pPsiA behaved as predicted under and without induction.
Principle
When pTacI is induced by IPTG, it drives the transcription of the PsiR gene coding for the PsiR protein which is predicted to be a transcription factor able to bind D-Psicose. If D-Psicose is present in the cell, the transcription factor will bind preferentially to it and thus be inactivated. The repression of the related promoter pPsi will be released, enabling the transcription of a fluorescent protein, mCherry. If D-Psicose isn’t present in the cell, PsiR will bind to pPsi, 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 (almost 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-Psicose concentration between 1mM 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-Psicose concentration is above 10mM, a 5 hour incubation after induction would give relevant results.
Basal expression
The biosensor show a basal expression of ….. arbitrary units of fluorescence at 9 hour post-induction. This basal activity even without psicose in the media is due to an imbalance between the amount of PsiR transcription factor available and the pPsiA promoter strength. Even when PsiR 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 xxx mM to xxx mM. This gives us two important pieces of information:
- First, the results show that PsiR seems to interact with psicose and therefore behaves as predicted. The same observation applies for the pPsiA promoter which seems tightly regulated by PsiR under psicose induction, behaving also as predicted.
- Second, the dynamic range of this biosensor appears to go from mM to mM psicose. This means that we will be able to use it in real applications for our bioscreening protocol to assess the production of psicose that could range from 1 mM to maximum 300 mM.
Figure 2. In vivo characterization of this Psicose Biosensor in E. coli DH5alpha. The graph shows the mCherry measured florescence over psicose concentration in the media. Each data point is the mean of number technical duplicates and of number biological triplicates. Error bars represent standard deviations.
Influence of fructose
Testing our psicose biosensor under real application conditions is vital. Indeed, we aim to measure the concentration of psicose in high fructose level media to observe the conversion of fructose into its epimer, D-Psicose by the D-Psicose 3-epimerase. To achieve this, our biosensor, and especially our transcription factor, has to be highly specific to psicose, in order to produce mCherry proportionally only to the concentration of D-Psicose.
We can see in the graph presented in Figure 3 that fluorescence intensity and fructose concentration are not correlated. Thus fructose doesn’t seem to influence the biosensor behavior since mCherry production isn’t a function of fructose concentration in the media. This finding implies that our transcription factor doesn’t bind to fructose and that our biosensor can be used in high fructose level media to measure psicose concentration. Therefore, this biosensor is suitable for assessing the activity of D-Psicose-3-Epimerase converting fructose into psicose.
Figure 3. In vivo characterization of this Psicose Biosensor in E. coli DH5alpha. The graph shows that mCherry measured florescence over fructose concentration in the media. Each data point is the mean of number technical duplicates and of number biological triplicates. Error bars represent standard deviations.
Influence of IPTG
We assessed for this biosensor the effect of IPTG on the fluorescence for a fixed concentration in psicose (Figure 4). The outcome is that IPTG, regardless of its concentration, doesn’t significantly influence the production of mCherry. It means that the PsiR amount in the cell is constant. Our hypothesis is that this phenomenon is due to the pTacI promoter leakiness, leading to a steady production of PsiR. Since our biosensor is carried by a high copy plasmid (pSB1C3), the number of lacO sites from pTacI is much bigger than the number of LacI molecules available in the cell. Even if this finding isn’t an issue for our experiments, this has to be considered. This issue can be addressed by performing the characterization of this biosensor in our improved backbone, the pSB1C3_LacIq plasmid (BBa_K2448038), that we designed exactly for this type of situation. pSB1C3_LacIq plasmid (BBa_K2448038) has a built-in LacI coding sequence under the control of a mutated version of its own natural promoter known as LacIq which leads to a 10-fold increase in LacI expression compared to the natural promoter.
Figure 4. Graph of the measure of florescence over fixed psicose concentration (1mM) in the media with different concentration of IPTG.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1337
Illegal XhoI site found at 62 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 104
Illegal AgeI site found at 791
Illegal AgeI site found at 2351
Illegal AgeI site found at 2463 - 1000COMPATIBLE WITH RFC[1000]
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