Part:BBa_K5127010
pBrer-Tes4
This composite part combines the BreR transcriptional regulator (BBa_K5127002) and the Tes4 gene (BBa_K3838613) that codes for butyrate.
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 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 to find out if there is any butyrate produced.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1012
Illegal EcoRI site found at 1024
Illegal XbaI site found at 775
Illegal XbaI site found at 1087
Illegal SpeI site found at 750
Illegal PstI site found at 345
Illegal PstI site found at 1345 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1012
Illegal EcoRI site found at 1024
Illegal NheI site found at 781
Illegal NheI site found at 804
Illegal NheI site found at 1058
Illegal NheI site found at 1081
Illegal SpeI site found at 750
Illegal PstI site found at 345
Illegal PstI site found at 1345 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1012
Illegal EcoRI site found at 1024
Illegal BglII site found at 1316
Illegal BamHI site found at 1018
Illegal BamHI site found at 1030
Illegal BamHI site found at 1615
Illegal XhoI site found at 1624 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1012
Illegal EcoRI site found at 1024
Illegal XbaI site found at 775
Illegal XbaI site found at 1087
Illegal SpeI site found at 750
Illegal PstI site found at 345
Illegal PstI site found at 1345 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1012
Illegal EcoRI site found at 1024
Illegal XbaI site found at 775
Illegal XbaI site found at 1087
Illegal SpeI site found at 750
Illegal PstI site found at 345
Illegal PstI site found at 1345 - 1000COMPATIBLE WITH RFC[1000]
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