| BNDS-China - iGEM 2024

Food Detection Module

Design and Construction of VFA0359-pDCA

In our platform design, DCA was adopted as food intake signal. To verify the inducibility of the DCA promoter (pVFA0359) in the presence of constitutive VFA0359 expression driven by apFAB254, GFP was inserted downstream of pVFA0359 as a reporter gene, which gives the overall plasmid named VFA0359-pDCA (Figure 1).

Figure 1. Plasmid design of VFA0359-pDCA. Created by biorender.com.

The VFA0359, apFAB254, and pVFA0359 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).

Figure 2. The AGE result of the PCR products of VFA0359-pDCA construction. A, materials to construct pDCA. B, golden gate assembly result of VFA0359-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 VFA0359-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.

Characterization of DCA biosensor using pBreR

We developed an alternative design for DCA detection. In this device, DCA inhibits the ability of the constitutively expressed repressor BreR to regulate the transcriptional activity of pBreR, therefore leading to the expression of downstream reporter gene. (Figure 4).

Figure 4. Plasmid design of pBreR plasmid. Created by biorender.com.

We used Golden Gate Assembly to construct pBreR plasmid. PCR and HGel Electrophoresis were performed to verify the materials of the assembly (Figure 5).

Figure 5. The AGE result of the PCR products of materials to construct pBreR plasmid.

To evaluate the inducibility of the downstream gene, we used GFP as the reporter gene to monitor promoter activity. 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 can detect DCA. (Figure 6).

Figure 6. Observation of pBreR with DCA+ (left) or DCA- (right). The left bacteria culture with DCA treatment showed strong green fluorescence.

We quantified the relationship between DCA concentration and pBreR promoter activity. The dynamic range of BreR sensor system was about 6-fold with the maximum signal achieved at 500μM DCA; above 500μM, the change in promoter activity became subtle (Figure 7).

Figure 7. Quantitative relationship between DCA concentration and fluorescence level. The GFP expression was quantified by measuring fluorescence / ABS600.

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 8).

Figure 8. A kinetic assay for GFP expression was conducted over 18 hours using various DCA concentrations. GFP expression was quantified by measuring fluorescence / ABS600__.

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 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.

Butyrate Sensing and Production Module

Design and Construction of PpchA

The butyrate sensing device includes three promoters: J23101, PpchA, and pLam. The leucine-responsive regulatory protein Lrp was constitutively expressed as driven by J23101. Then, Lrp activates the expression of cI repressor in the presence of butyrate, and the cI protein inhibits promoter pLam activity (Figure 9). The pCI-Lam, PpchA, protein CI, and GFP were synthesized by Genscript. The Lrp protein was supported by NMU-China.

Figure 9. Plasmid design of PpchA. Created by biorender.com.

Characterization of butyrate biosensor using PpchA

Initially, our plasmid's RBS component, synthesized by Genscript, had an unwanted point mutation. But we did characterization to see whether it could still function well (Figure 10).

Figure 10. Kinetics of PpchA (with RBS point mutation) with multiple butyrate concentrations over 18 hours. Fluorescence / ABS600 was used to represent GFP expression; higher fluorescence represented lower PpchA activity.

The kinetics results of this error-containing plasmid did not achieved a desired performance: the dynamic range of this system was less than 1.5-fold at after added a gradient of butyrate concentration. Therefore, we've tried to fix the point mutation.

We used GoldenGate Assembly to construct PpchA-RBS-fixed. PCR and Gel Electrophoresis were performed to verify the success in constructing each component and the plasmid (Figure 11).

Figure 11. 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.

We again used kinetics to quantitatively test the effectiveness of the above design for butyrate detection. A gradient of butyrate concentration was added into E. coli transformed with PpchA, and the value of fluorescence / ABS600 over time was detected using the plate-reader to represent GFP expression levels (Figure 12).

Figure 12. Kinetics of PpchA (RBS fixed) with multiple butyrate concentrations over 16.7 hours. Fluorescence / ABS600 was used to represent GFP expression; higher fluorescence represented lower PpchA activity.

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.

Characterization of butyrate biosensor using pHpdH

In our plasmid design for pHpdH, butyrate activates pHpdH in the presence of the constitutively expressed hpdR (Figure 13).

Figure 13. Plasmid design of pHpdH. Created by biorender.com.

The hpdR, phpdH, and phpdR were synthesized from Gensript. We used Golden Gate Assembly to construct pHdpH. PCR and Gel Electrophoresis were performed to verify the success in constructing the fragment and backbone of the overall pHdpH plasmid (Figure 14).

Figure 14. The AGE result of the PCR products of pHdpH construction. A, materials to construct pHdpH. B, golden gate assembly result of pHdpH construction. The band at 4555bp in (B) indicated the success in plasmid construction.

We used kinetics to quantitatively test the effectiveness of this butyrate detection system. A gradient of butyrate concentration was added into E. coli transformed with pHpdH, and the value of fluorescence / ABS600 over time was detected using the plate-reader to represent GFP expression (Figure 15).

Figure 15. Kinetics of GFP expression over 16.7 hours. Fluorescence / ABS600 was used to represent GFP expression.

When the butyrate concentration was below 20mM, our system showed the desired trend, as fluorescence increases proportionally with butyrate concentration. Above this point, however, the system failed to distinguish between 20mM, 40mM, and 86mM butyrate. This might be because 20mM butyrate was sufficient to activate PhpdH to its highest activity. Since our goal was to detect the shortage of butyrate in the gut system, this result showed that our design was successful and achieved our goal.

Characterization of butyrate biosensor using pHpdH-CI

To integrate the butyrate biosensor into our ultimate project goal, we designed the CI gene into the pHpdH sensing system plasmid. In this system, when butyrate levels are low, the output of GFP will be higher (Figure 16). This design enables us to monitor and respond to butyrate concentration effectively within our system.

Figure 16. Plasmid design of HpdR-pHpdH-cI-GFP Created by biorender.com.

Figure 17. The AGE results of the PCR products of pHpdH-CI construction. The materials used to construct pHpdH-CI.

We require additional time to further characterize and evaluate the degradation function to ensure optimal performance and reliability in our system.

Verification of butyrate production by Tes4

In our plasmid design of Tes4, IPTG was added to induce the expression of Tes4 (Figure 18). The Tes4 DNA sequence and the vector pET-28a(+) were synthesized and obtained from Genscript.

Figure 18. Plasmid design of Tes4. Created by biorender.com.

To verify the successful expression of Tes4 and its effectiveness to produce butyrate, we performed SDS-PAGE to the proteome of E. coli expressing Tes4. The band at 19.5kDa represented the successful expression of Tes4 (Figure 19).

Figure 19. SDS-PAGE result of Tes4 production. Lane 1, Protein ladder. Lane 2, Tes4 plasmid with IPTG added. Lane 3, BL21 WT. Lane 4, Tes4 plasmid without IPTG added. Lane 6, Protein ladder.

We also performed GC-MS to verify our system's ability to produce butyrate by Tes4. The peak at 11.39 min in (D) suggested the successful production of butyrate (Figure 20).

Figure 20. GC-MS results of butyrate production. Peaks at about 11.4 min represented butyrate. A, pET28a(+)-Tes4 IPTG(-). B, pET28a(+)-Tes4 IPTG(+) without lysis. C, LB blank (Maybe contaminated). D, pET28a(+)-Tes4 IPTG(+) after lysis. E,1mmol/3mL butyrate standard solution. F, 10mmol/3mL butyrate standard solution. The peaks at 11.25-11.45 min indicated the presence of butyrate.

Tes4 Genome Integration Test

We wish to incorporate Tes4 enzyme encoding genes into the genome of our bacteria to lower its burden and maintain better ability of butyrate production. Thus, we developed a genome integration technique using Lambda Red recombinase, and tested it.

Phase I

The pRed-Aspink expresses the Lambda Red recombinase under the control of an arabinose-inducible promoter. The plasmid also constitutively expresses the pigment protein asPink, which serves as a visual marker to confirm the presence of the plasmid in the host cells.

Figure 21. Plasmid design of pRed-Aspink. Created by biorender.com.

The Lambda Red recombinase proteins was derived from BNDS-China previous plasmid stock, and Aspink protien was derived from 2023 iGEM distribution kit. We used Golden Gate Assembly to construct pRed-Aspink. PCR and Gel Electrophoresis were performed to verify the success in constructing the fragment and backbone of the full plasmid (Figure 22).

Figure 22. The Agarose gel electrophoresis result of the PCR products of pReplace construction. A,B, materials to construct pReplace. C, golden gate assembly result of pReplace construction. The band at 6910bp in (C) indicated the success in plasmid construction.

The plasmid was transformed into E. Coli MG1655, with the colonies displaying a pink color, indicating the successful construction and transformation of the plasmid.

Phase II

The second plasmid, pDual-Select, was designed to facilitate both positive and negative selection. Initially, chloramphenicol resistance is used for positive selection, enabling the identification of colonies with successful transformation of pDual-Select. Following recombination by the Lambda Red system, the expression of SacB protein allows for negative selection, as SacB is lethal in the presence of sucrose. This step ensures that only colonies with the correct integration of the gene of interest into the genome survive.

Figure 23. Plasmid design of pDual-Select. Created by biorender.com.

The sequence of homology arms and R6K Ori were obtained from BNDS-China's previous plamids. We used Golden Gate Assembly to construct pReplace. PCR and Gel Electrophoresis were performed to verify the success in constructing the fragment and backbone of the full plasmid (Figure 24).

Figure 24. The Agarose gel electrophoresis result of the PCR products of pReplace construction. A,materials to construct pReplace. B, golden gate assembly result of pReplace construction. The band at 5831bp in (B) indicated the success in plasmid construction.

The plasmid was transformed into E. Coli Trelief 5-alpha for contruction, by plating them on the Kanamycin plates, the construction of this plasmid was being confirmed.

At the same time, the phase I cells were prepared as chemically competnet cells and induced with arabinose for a 3-hour resuscitation. The pDual-Select plasmid was then transformed into these Phase I competent cells, which were subsequently plated on chloramphenicol plates for selection. Only bacteria with successful genome integration of the plasmid will grow on the plate, due to the lost of the second plasmid (Figure 25).

Figure 25. The plating result of phase II cells.

Phase III

The third plasmid we constructed was designed to facilitate the integration of the Gene of Interest (GOI) into the genome. This plasmid includes a barcode for colony PCR verification post-integration.

Figure 26. Plasmid design of pReplace.

Figure 27. The Agarose gel electrophoresis result of the PCR products of pReplace construction. A, B, materials to construct pReplace. C, golden gate assembly result of pReplace construction. The band at 2932bp in (C) indicated the success in plasmid construction.

After transforming pReplace into Phase II competent cells, we initially observed a bacterial lawn on the plate (Figure 28A). After incubating the plate at 37 degree Celcius for three days, we identified single bacterial colony (Figure 28B). Then, we inoculated colonies and performed colony PCR, which showed the correct length, indicating successful integration (Figure 28C). However, due to limitations in sequencing capabilities in our lab, we were unable to obtain sequencing results for successful PCR products.

Figure 28. Observation and PCR verification of Phase III cells. A, The bacterial lawn of Phase III cells. B, The single colonies occur after being cultured at 37 degree Celsius for several days. C, The AGE result for Phase III colony PCR verification. The band at 1753bp indicates a successful amplification from genome.

Coupling the butyrate bioproduction to food detection

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 29).

Figure 29. Plasmid design of BreR-Tes4. Created by biorender.com.

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 30).

Figure 30. The AGE results of the PCR products of BreR construction. A and B, materials to construct BreR-Tes4. C, Golden gate assembly result of BreR-Tes4 construction. The band at 4372bp in (C) indicated the success in plasmid construction.

We performed SDS-PAGE to verify the presence of Tes4. The band representing Tes4 (19.5kDa) was thicker in the DCA-treated group, showing the successful induction of Tes4 by DCA (Figure 31).

Figure 31. The SDS-PAGE result of successful Tes4 expression induced by DCA.

We are trying to further test the BreR-Tes4 plasmid with GC-MS assay to find out if there is any butyrate produced.

IAA Sensing and Degradation Module

Characterization of IAA biosensor

IAA was detected through its inducibility to the promoter pIacR. In our plasmid design, IAA activates pIacR in the presence of constitutively expressed iacR, therefore inducing the expression of the downstream gene (Figure 32).

Figure 32. Plasmid design of pIacR. Created by biorender.com.

The iacR and PiacR that were used in this plasmid were synthesized by Genscript. We used kinetics to quantitatively test the effectiveness of IAA detection using the designed system above. A gradient of IAA concentration, from 0μM to 100μM was added into E. coli transformed with pIacR, and the value of fluorescence / ABS600 over time was detected using the microplate reader to represent GFP expression (Figure 33).

Figure 33. Kinetics of pIacR over 16.7 hours. Fluorescence / ABS600 was used to represent GFP expression. The darker blue color, the higher IAA concentration. The numbers represent the concentration (in μM) of IAA added.

After cultured at 37 ℃ for 18 hrs, the normative fluorescence/ABS 600 values increased as IAA concentration increased, showing the successful design of the IAA detection module.

Verification of IAA degradation

In the IAA degradation module, we used IPTG to induce the expression of iadC, iadD, and iadE. They function together to degrade IAA (Figure 34).

Figure 34. Plasmid design of PiadCDE. Created by biorender.com.

The iadC, iadD, and iadE were synthesized by Genscript. To verify the successful expression of iadC, iadD, and iadE, we performed PAGE to the expressed protein of E. coli transformed with PiadCDE with or without IPTG added. The bands at indicated lengths showed the successful expression of iadCDE (Figure 35).

Figure 35. SDS-PAGE result of iadCDE expression. Lane 1, 6.5-200kDa protein ladder. Lane 2-4, PiadCDE with IPTG(+). Lane 5, PiadCDE with IPTG(-). Lane 6, 6.5-200kDa protein ladder. Lane 7-8 PiadCDE with IPTG(-).

To verify the effectiveness of iadCDE degrading IAA, we used the Salkowski reagent to quantitatively detect the amount of IAA (Gordon et al, 1951). First, we constructed an IAA standard curve to show the feasibility of using the reagent to detect IAA. A gradient of IAA was mixed with Salkowski reagent (see our protocols) and waited for 1 hour for color formation. The absorbance at 530 nm was measured to indicate the IAA concentration, which shows an approximately linear relationship and thus validated the effectiveness of the Salkowski reagent in IAA concentration measurement (Figure 36).

Figure 36. IAA standard curve.

After ensuring the usefulness of the reagent, we cultured transformed bacteria together with IAA and made an IAA degradation curve. After induction, the group with PiadCDE but without IPTG showed higher IAA concentration than BL21 WT, while the group with PiadCDE with IPTG added showed a much lower amount of IAA compared to BL21 WT (Figure 37). This result showed our system could successfully degrade IAA by expressing iadCDE.

Figure 37. IAA degradation curve. Black, BL21 WT. Red, E. coli transformed with PiadCDE with IPTG added. Blue, E. coli transformed with PiadCDE without IPTG added.

Verification of J23119-iadCDE

To optimize the IAA degradation efficiency, we constructed a second plasmid that replaces the inducible T7 promoter with the constitutive promoter J23119. This modification ensures continuous expression of the IAA degradation pathway, potentially enhancing its overall effectiveness.

Figure 38. Plasmid design of J23119-iadCDE. Created by biorender.com.

Figure 39. The AGE results of the PCR products of J23119-iadCDE construction. A, materials to construct J23119-iadCDE. B, Golden gate assembly result of J23119-iadCDE construction. The band at 7947bp in (B) indicated the success in plasmid construction.