Difference between revisions of "Part:BBa K5127001"

Revision as of 09:45, 2 October 2024

pVFA0359

This part is the promoter regulated by repressor VFA0359.

Usage and Biology

VFA0359 is a TetR-family repressor responsive to deoxycholic acid (DCA) from Vibrio fischeri. We choose to test this because it has been shown to have low basal activity and 440-fold dynamic range in B. thetaiotaomicron upon induction by DCA (Taketani et al., 2020), so it's a promising choice for our E. coli probiotic platform for bile acid biosensing. When DCA is present, VFA0359 binds to DCA and releases the operator DNA, thus enabling downstream gene expression.

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 VFA0359 works as an effective food sensor for detecting DCA.

Design and Construction of pDCA

This system has not been tested in E. coli previously and shows problems such as undesired offsite transcription when expressed in E. coli as predicted by the Promoter Calculator (LaFleur et al., 2022). See our enginering success page for more details (https://2024.igem.wiki/bnds-china/engineering).

Considering that VFA0359 is a TetR-family repressor, which shares homology with the previously reported "jungle express" system by EilR (Ruegg et al., 2018), we modified the jungle express system by replacing the EilR coding frame and EilO operator with VFA0359 CDS and the predicted operator sequences respectively. In our design, the expression of repressor VFA0359 is driven by a constitutive promoter apFAB254 (Mutalik et al., 2013). For the regulated promoter pVFA0359, we first predicted the palindromic operator sequence by SnowPrint (d’Oelsnitz et al., 2024) and inserted two operators in the phage early promoter used by jungle expression system: one overlaps the -35 and -10 hexamer and the other one downstream -10. As for the reporter gene, we inserted a GFP downstream pVFA0359, forming a complete biosensor (Figure 1).

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

The individual parts VFA0359, apFAB254, and pVFA0359 shown above were synthesized by Genescript. We used Golden Gate Assembly assay to assemble the full plasmid, pDCA. PCR and Gel Electrophoresis were performed to verify the success in the fragment and backbone of the overall pDCA plasmid (Figure 2). We did a series of optimization for this part to achieve the desired result. See our enginering success page for more details (https://2024.igem.wiki/bnds-china/engineering).

Figure 2. The AGE result of the PCR products of pDCA construction. A, materials to construct pDCA. B, golden gate assembly result of pDCA construction. The band at 4544bp in (B) indicated the success in plasmid construction.

Characterization of DCA biosensor using pDCA

To assess DCA's inducibility on downstream genes, we quantified the relationship between DCA concentration and pVFA0359 activity. A gradient of DCA concentration was added to bacterial cultures, and the fluorescence / ABS600 values were measured over time using a plate-reader to assess promoter activity. The fluorescence exhibited concentration-dependent behavior. As the concentration of DCA in the medium increased, higher expression of GFP was induced in the bacteria (Figure 3).

Figure 3. A kinetics assay for pDCA was conducted over 18 hours using various DCA concentrations. GFP expression was quantified by measuring fluorescence / ABS600. Black, 1000μM DCA. Purple, 500μM DCA. Blue, 250μM DCA. Cyan, 125μM DCA. Green, 62.5μM DCA. Yellow, 31.25μM DCA. Orange, no DCA added.

From this, it can be concluded that the activity of the pVFA0359 promoter can be induced by bile acid in E. coli, which demonstrated the biosensing capacity of this construct.

Nevertheless, it was also observed that the output in normalized fluorescence level at the highest induction concentration, 1000µM bile acid, was still low. This suggests a limited dynamic range for this biosensor. Thus, our team constructed an alternative DCA sensor for further investigation.

Sequence and Features