Difference between revisions of "Part:BBa K4442001"

Latest revision as of 06:34, 2 October 2024

The butyrate sensing system PpchA-PLEE1 originates from the enterohemorrhagic Escherichia coli strain O157:H7 which was discovered by Nakanishi et al. (2009). The system below includes the sequences of the pchA promoter and pchA regulator, located upstream, as well as the LEE1 promoter and RBS downstream. A complex of the leucine-responsive regulatory protein and butyrate activates the system. To add an fluorescent output or any other sort of output protein, simply add the base pairs downstream of the RBS.

Nakanishi et al.1 found that the leucine-responsive regulatory protein (Lrp) forms a complex with butyrate which after binding to the promoter of pchA, activates the transcription of the pchA regulator. The pchA regulator then binds to the promoter of LEE1, to transcribe the ler gene. In Bai & Mansell’s2 paper, the ler gene was replaced with GFP to report the sensing of butyrate. Similarly, in our project, the pchA regulator would bind to the promoter of LEE1 but instead express YFP.

More information can be obtained here: Nakanishi N, Tashiro K, Kuhara S, Hayashi T, Sugimoto N, Tobe T. Regulation of virulence by butyrate sensing in enterohaemorrhagic escherichia coli. Microbiology. 2009;155(2):521–30. Bai Y, Mansell TJ. Production and sensing of butyrate in a probiotic E. coli strain. International Journal of Molecular Sciences. 2020;21(10):3615.

A decrease in butyrate concentration in fecal samples was found to correlate with depression. As part of our goal to create a microbial sensor to aide in the diagnosis of depression, this system will allow us to detect the levels of butyrate through fluorescence intensity.

The pchA promoter, pchA, and LEE1 promoter sequences were all obtained from the original study authors, Yanfen Bai and Thomas J. Mansell.

NMU_China_2023

Correction

The team got the wrong order of the composite sequence in mistakes, which should be Ppcha-pcha-plee1 actually.

Short description

The Butyrate Sensing System can "sense" the level of butyrate, its core event is the binding of the Lrp-butyrate dimer complex to the promoter PpchA. Since the activation of promoter PpchA can promote downstream gene expression, the volume of downstream products can reflect the perceived butyrate concentration.

Our validation via experiments

Our team has made the following additions to the development and validation of the Butyrate Sensing System:

1. We designed two gene segments: PpchA-pchA-PLEE1-EGFP and PpchA-pchA-EGFP. After characterization, we measured and compared fluorescence intensity and bacterial growth curves, proved that the segment involving plee promoter (i.e. PpchA-pchA-PLEE1-EGFP) has a higher sensitivity to butyrate as well as having a stronger ability to enhance downstream gene expression.

2. We demonstrated in controlled experiments that increased Lrp expression can indeed enhance the sensitivity of PpchA to butyrate.

3. We tested the capacity for "sense" of the designed gene segment PpchA-pchA-PLEE1-EGFP, and found that the higher the concentration of butyrate, the stronger the fluorescence, which proved that our engineered bacteria could indeed sensitively sense the increase of butyrate concentration. Then, we designed the gene segment PpchA-pchA-PLEE1-CI-Plam-EGFP, in which CI protein was used to bind promoter Plam and inhibit downstream EGFP expression. The experiment found that the higher the concentration of butyrate, the more obvious the inhibition of fluorescence expression (indicating that the more CI protein produced by butyrate). This confirmed once again that our engineered bacteria could indeed sensitively sense an increase in butyrate concentration. Overall, we validated the feasibility of butyrate receptors.

Fluorescence Intensity(OD480/OD600) with 0mM, 10mM and 20mM butyrate cocultured in PpchA-pchA-PLEE1-EGFP engineered bacteria.

a.Fluorescence observation of PpchA-pchA-PLEE1-EGFP engineered bacteria fluid cocultured with different concentrations of butyrate through fluorescence microscopic

b.Fluorescence intensity of PpchA-pchA-PLEE1-EGFP engineered bacteria fluid cocultured with different concentrations of butyrate

a.Fluorescence observation of PpchA-pchA-PLEE1-CI-Plam-EGFP engineered bacteria fluid cocultured with different concentrations of butyrate through fluorescence microscopic

b.Fluorescence intensity of PpchA-pchA-PLEE1-CI-Plam-EGFP engineered bacteria fluid cocultured with different concentrations of butyrate

Team: BNDS-China 2024

We wish to build a butyrate biosensor in our project. In our design, leucine-responsive regulatory protein (Lrp) is constitutively expressed using a constitutively expressed promoter, pJ23101, while CI protein is regulated by the butyrate sensor's control and binds to the pLam promoter to inhibit the expression of downstream protein. When butyrate is not present, the expression of CI repression is not activated, and hence the target gene downstream pLam promoter will be expressed. In the presence of butyrate, Lrp forms a complex with butyrate and then binds to the pPcha promoter to activate the expression of the CI repressor, which then inhibits promoter pLam, and stops the expression of the target gene (Figure 1).

Figure 1. Plasmid design of PpchA. Created by biorender.com.

Characterization of butyrate biosensor using PpchA

The plasmid synthesized by the company contained a point mutation on the ribosome binding site (RBS), resulting a low translational rate for Lrp. To address this, we utilized the promoter calculator function by De novo DNA (LaFleur et al., 2022). The results confirmed that Lrp expression was significantly below our expectations (Figure 2).

Figure 2. Promoter calculator function with mutated and fixed RBS. A, the translation rate of our design without the RBS point mutation. B, the decreased translation rate affected by the RBS point mutation.

In the wet lab, we performed a kinetics analysis of this plasmid, which revealed slight differences and a correct trend as the gradient of inducer was added, though the dynamic range was suboptimal (Figure 3).

Figure 3. Kinetics of PpchA before fixing with multiple butyrate concentrations over 16.7 hours. Fluorescence / ABS was used to represent GFP expression; higher fluorescence represented lower PpchA activity. The butyrate concentration ranges from 0mM to 70mM.

We wish to fix this RBS point mutation to gain better results. The two fragments for goldengate is based on the sysnthesized plasmid of pUC57-PpchA-CI. Goldengate Assembly was used to construct the plasmid (Figure 4). Sequencing verified the construct as correct.

Figure 4. The AGE results of the PCR products of PpchA construction. A, materials to construct PpchA. B, Goldengate assembly result of PpchA construction. The band at 5278bp in (B) indicated the success in plasmid construction.

Following the successful construction of the PpchA-RBS-Fixed plasmid, we conducted a kinetics analysis to evaluate its performance (Figure 5).

Figure 5. Kinetics of PpchA after RBS was fixed with multiple butyrate concentrations over 16.7 hours. Normative fluorescence / ABS600 values were used to represent GFP expression; higher fluorescence represented lower PpchA activity. The butyrate concentration ranges from 0 to 100 mM.

The overall performance of the PpchA-Lrp sensor is sufficient to demonstrate its ability to detect and respond to the presence of butyrate accurately at most butyrate concentrations. However, the sensor exhibits low sensitivity in distinguishing between small variations in butyrate levels; furthermore, the dynamic range of this detection system was only about 2-fold. This is likely attributed to the inherent limitation of this system, as a similar noisy butyrate induction pattern of PpchA-Lrp has been previously reported in literature (Serebrinsky-Duek et al., 2023). To address those limitations, we developed an alternative sensor, pHdpH, for further investigation.

References

LaFleur, T. L., Hossain, A., & Salis, H. M. (2022). Automated model-predictive design of synthetic promoters to control transcriptional profiles in bacteria. Nature Communications, 13(1). https://doi.org/10.1038/s41467-022-32829-5

Kineret Serebrinsky-Duek, Barra, M., Danino, T., & Garrido, D. (2023). Engineered Bacteria for Short-Chain-Fatty-Acid-Repressed Expression of Biotherapeutic Molecules. Microbiology Spectrum, 11(2). https://doi.org/10.1128/spectrum.00049-23