Difference between revisions of "Part:BBa K5127002"

Latest revision as of 13:06, 2 October 2024

BreR

This part is a coding sequence for BreR.

Usage and Biology

BreR is an allosteric TetR-family transcription factor responsive to deoxycholic acid (DCA) identified in Vibrio cholorae. We choose to test this as well because it has been validated in E. coli, with an about 230-fold dynamic range to DCA (Beabout et al., 2023), so it's a promising choice for our E. coli probiotic platform. As a transcription factor, BreR plays a significant role in bile acid sensing by demonstrating a tight long-term binding to the promotor. In the BreR system, BreR is constitutively expressed and binds to its corresponding promoter pBreR which contains the operator sequence. When DCA is present, it binds to BreR, releasing it from the operator and triggering the expression of downstream genes.

Team: BNDS-China 2024

Our team aims to detect the intake of food before activating our comprehensive platform that regulates levels of gut metabolites, with DCA (a subtype of bile acid) as the indicator of food presence. Within this framework, the BreR system stands out as an effective food sensor for detecting DCA, and the response range best aligns with the fluctuations of DCA levels in human intestines.

Structural analysis of BreR repressor

As expected, BreR structure predicted by alphafold 3 (Abramson et al., 2024) suggests a homodimer oligomer (Figure 1A). Moreover, the protein-DNA interaction prediction by alphafold 3 accurately predicted the BreR binding to the BreO operator surrounded by the upstream spacer and downstream promoter DNA (Figure 1B). Structural alignment by PyMOL cealign function showed a significant structural shift upon DNA binding (Figure 1C, red circled). Taken together, the structural evidence from alphafold 3 further the DNA binding capability of BreR, and we thus adopted this allosteric TetR-family transcription factor for DCA biosensor design.

Characterization of DCA biosensor using BreR

In our design, we used constitutive promoter J23106 for BreR expression. The pBreR is designed with an optimized version of the J23101 promoter with BreO operator incorporated (Beabout et al., 2023) (Figure 2). We inserted GFP downstream to pBreR as the reporter gene.

We used Golden Gate Assembly to construct pBreR plasmid. PCR and Gel Electrophoresis were performed to verify the materials of the assembly (Figure 3).

To evaluate the inducibility of the downstream gene, two cultures of E. coli transformed with the BreR plasmid were prepared, with DCA added to one. The DCA-treated culture exhibited strong green fluorescence, qualitatively demonstrating that our design is responsive to DCA (Figure 4).

We quantified the relationship between DCA concentration and pBreR promoter activity. The dynamic range of BreR biosensor system was about 6-fold with the maximum signal achieved at 500μM DCA; above 500μM, the change in promoter activity became subtle (Figure 5).

Further, we characterized the kinetic behavior of this biosensor. A gradient of DCA concentration was added into bacterial cultures, and fluorescence / ABS600 values were measured over time using a plate-reader to assess promoter activity (Figure 6).

The results aligned with our expectations, in which bacteria cultures added with higher DCA concentration had stronger GFP expression levels. The dynamic range of BreR sensor system was about 5-fold at 1000μM DCA, much higher compared to that of pDCA_VFA0359 system (about 2-fold).

This trend was especially clear when DCA concentration was above 62.5μM. When DCA concentration was below 62.5μM, the difference in DCA concentration between groups might be too small to be reflected in significant difference in GFP expression.

Applying DCA biosensor for butyrate production

To implement our overall design, we placed Tes4 downstream to promoter pBreR to make the butyrate production responsive to the food availability. In this way, the presence of bile acid will trigger the expression of Tes4 and produce butyrate. We replaced the GFP reporter gene in the BreR-mediated DCA biosensor with the coding frame of Tes4 (Figure 7).

We used Golden Gate Assembly to construct BreR-Tes4 from BreR backbone and Tes4 fragment. PCR and Gel Electrophoresis were performed to verify the success in constructing each component and the full plasmid (Figure 8).

We performed SDS-PAGE to verify the expression of Tes4. The band representing Tes4 (19.5kDa) was thicker in the DCA-treated group, showing the successful induction of Tes4 by DCA (Figure 9).

We are trying to further validate the production of butyrate with GC-MS assay.

Sequence and Features