1. BSS, FXR-ddRFPA1 and RXR-ddRFPB1 parts construction
In accordance with the iGEM safety policy, we synthesized the BSS, FXR and RXRα genes. The ddRFPA1 and ddRFPB1 genes were kindly given to us by Prof. Josef Voglmeir. We recombined these 5 genes, pUC57 and pET-29a(+) plasmid backbones by homologous recombination , respectively and compositely(Figure 1).
Figure 1. Plasmid maps. a. Constitutive expression of BSS, BBa_K4164008; b. Constitutive expression of FXR-ddRFPA1 and RXR-ddRFPB1, BBa_K4164011; c. Inductive expression of RXR-ddRFPA1, BBa_K4164015; d. Inductive expression of FXR-ddRFPA1, BBa_K4164024; e. Inductive expression of BSS, BBa_K4164013; f. Inductive expression of FXR-ddRFPA1-RXR-ddRFPB1, BBa_K4164017.
2. Feasibility verification of ddRFPA1-ddRFPB1
In order to verify the interaction between two monomeric proteins, ddRFPA1 and ddRFPB1, we connected ddRFPA1 and ddRFPB1 by a flexible linker(3*GGGGS). We cloned this part into the pET-29a(+)(Figure 2a) and expressed recombinant proteins in E. coli BL21(DE3).
The target bacterial inoculum was streaked on one LB agar plate supplemented with 1 mM IPTG. Following overnight incubation at 37℃, we can see the bright red fluorescence (Figure 2c and d). In this case, we chose ddRFPA1 and ddRFPB1 as our report device.
Figure 2. a. Results for pET29a(+)-ddRFPA1-B1. Lane2, lane 4, the whole length of these plasmids is 6594bp. b. The plasmid map of pET29a(+)-ddRFPA1-B1. c, d. Fluorescence image of E. coli expressing ddRFPA1-ddRFPB1 and control.
3. Effects of CDCA concentration and promoter intensity on report device within the cell
On the basis of parts above, we constructed FXR-ddRFPA1 and RXR-ddRFPB1 expression plasmids with different promoters(BBa_J23105, T7lac promoter). We cultured them in LB medium with antibiotics and grew at 37°C until OD600 was about 0.6. Then IPTG was added to the final concentration of 0.5mM and induced protein expression at 28℃ for 3 hours. We tested fluorescence intensity at different concentrations of CDCA (0, 10, 25, 50, 100μM) in E. coli BL21(DE3). Samples were taken after incubation at room temperature for eight hours. Fig. 3 shows the effects of CDCA concentration and promoter intensity on different plasmids within the cell.
Figure 3. Effects of CDCA concentration and promoter intensity on report device within the cell.
It is clear that at a CDCA concentration of 25μM, the T7lac promoter has the best results. Therefore, we chose T7lac promoter to form our final detection device.
4. Purification of FXR-ddRFPA1 and RXRα-ddRFPB1
While the T7lac promoter has better sensitivity relative to J23105(low intensity promoter), the difference in fluorescence intensity is not too significant when the CDCA concentration is 10μM or 25μM from Figure 3. Considering the high-level transcription of the gene in the presence of T7 RNA polymerase, which may result in the formation of inclusion bodies, we tried to determine the solubility of our proteins by purifying 6xHis-tagged proteins under native conditions and performing SDS-PAGE. Compare the abundance of the band with the size of interest.
Figure 4. Purification of FXR-ddRFPA1 and RXR-ddRFPB1.Lane 1: protein contained in the pellet after bacterial disruption. Lane 2: RXR-ddRFPB1 purified from supernatant of bacteria liquid. Lane 3: FXR-ddRFPA1 purified from supernatant of bacteria liquid in optimized expression conditon.
As shown in the Figure 4, RXR-ddRFPB1(78.9kDa) is soluble and can be purified by Ni-NTA affinity chromatography, while most of the FXR-ddRFPA1 is in the pellet after lysis and can't be purified. The insolubility of FXR-ddRFPA1(83.0kDa) might be caused by the formation of inclusion bodies, so we induced expression at a lower temperature and reduced the induction time to optimize the expression conditions. Figure 4 shows that inducing protein expression at 16℃ overnight can increase the portion of soluble FXR-ddRFPA1.
5. Implementation and debugging of cell-free system
To reduce the possibility of inclusion body formation and simplify the test process, we designed a cell-free system composed of two tubes.
The first tube included the substrate processing device which uses bile acid sulfate sulfatase to hydrolyze the 3-sulfate esters of bile acid. We used the methods described by Y. TAZUKE et al. (1992) to assay sulfatase activity. Figure 5 shows that the activity of BSS used was estimated at approximately 0.15 unit.
Figure 5. Assay of sulfatase activity. A reaction mixture containing 1.0 mL of 100 mM Tris buffer(pH8.0), 0.20 mL of 15mM [β-NAD, 0.20mL of 1mM CDCA-S, and 1.45mL of distilled water in eppendorf tube was incubated at 30°C for 10min, and then added 0.1mL of the cell-free solution and 0.05mL of β-HSD solution(10 units/mL).
The second tube included a detection device and a report device. We added the corresponding plasmids to the cell-free system, and then incubated them to a sufficient concentration of proteins. Then we added different concentrations of CDCA(0, 10, 25, 50, 100μM) to test the cell-free system. After 8 hours, we could see a clear difference in fluorescence intensity under the corresponding wavelength of light excitation.
Figure 6. Impact of different concentrations of CDCA on report device fluorescence intensity. blank: cell-free system with no plasmid a. Direct observation of fluorescence. b. Fluorescence under specific wavelength excitation.
From Figure 6, we also verified that a 5-fold dilution of urine samples can help us better distinguish between patients and healthy ones, in which case the CDCA concentration of healthy people failed to show visible fluorescence and the patients' urine did the opposite.
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