Overview
Our liver disease test kits provide a rapid and convenient home chronic hepatitis testing method based on bile acid levels.
Considering the inconvenience and high cost of hospital testing, we use synthetic biology to design gene circuits, constructing the biosensor with three devices: substrate processing device, detection device and report device.
In addition, to make our system more suitable for home testing, we try to develop a cell free system and make a test kit with corresponding hardware (Hardware).
Substrate Processing Device
Bile acids from urina sanguinis are regarded as the testing substrate of our system. Because compared to the serum total bile acid, the urinary bile acid is hardly affected by diet. Meanwhile, with the convenient sampling, it is an ideal index for daily detection.
In urine, bile acids are mainly chenodeoxycholic acid (CDCA), deoxycholic acid (DCA) and other bile acids, which account for more than 50% of the total bile acids. However, 70~80% of bile acids in urine are sulfonated bile acids, which are difficult for our receptor to capture. Therefore, we need desulfation to meet our needs.
Bss is the sulfatase from Pseudomonas testosteroni. It can remove the 3-sulphate sulfate groups of cholic acids (CADC, DCA, CA and so on), which are the predominant modification of sulfonated bile acids in urine. Further, with BSS, the average analytical recovery of various sulfonated bile acids in urine is 98%, which makes it a highly efficient and ideal enzyme for our project.
Figure 1. BSS desulfates on sulfonated
Detection Device
Our detection device consists of Farnesoid X Receptor (FXR) and Retinoid X Receptor (RXR).
FXR can efficiently identify and bind with CDCA, Lithocholic Acid (LCA) and DCA. Once activated by bile acids, it undergoes a conformational change. Especially for CDCA, FXR has the strongest recognition effect, with a half-effective concentration of about 10μM.
In human body, the activated FXR undergoes a conformational change and dimerizes with RXR. Then, they act together on the promoter to open the expression of downstream genes to regulate some metabolic pathways. On this basis, we simultaneously introduce them into our chassis as the detection device. When activated by de-sulfated CDCA, the above response will be triggered, driving the downstream report device.
Figure 2. FXR activates IBABP by binding directly to the IBABP promoter as a FXR/RXR heterodimer.
Report Device
For some reasons (Engineering LOOP 1 and 2), we choose dimerization-dependent fluorescent protein(ddRFP) as our reporter. This protein introduces an interface breaking mutation into dTomato. Furthermore, the least autodimerized and the most responsive parts are selected through directed evolution. It is formed by the polymerization of two monomeric proteins, ddRFP-A1 and ddRFP-B1. When they are in their monomeric states respectively, ddRFP-A1 shows weak fluorescence, while ddRFP-B1 shows no fluorescence. By the time the two parts dimerize, this molecule shows a red fluorescent signal visible to the naked eye.
We respectively connect ddRFP-A1 and ddRFP-B1 to FXR and RXR through linker. Based on this, when FXR and RXR dimerize, the downstream ddRFP-A1 and ddRFP-B1 are correspondingly driven to dimerize and emit fluorescence.
Figure 3. Structural model of ddRFP-A1B1
Gene Circuit
On this basis, we optimized the codons of above genes to make them suitable for expression in our chassis organism Escherichia coli BL21. We hope that under the joint action of these components, our chassis E. coli BL21 can run the whole system perfectly, laying foundation for the cell-free system.
Figure 4. Gene circuit
Cell Free System
With reference to the literature and the work of the iGEM team in previous years, we construct our own cell free reaction platform (Protocol), serving the purpose of homely rapid detection.
To prepare the reaction system, we first lyophilized the two plasmids of three devices and the cell-free system. Then, mix the substrate processing device and other devices with the cell-free system respectively. When a certain concentration of urine (bile acid) is added, the above-mentioned series of reactions occur rapidly. After incubation for some time, ddRFP-A1 and ddRFP-B1 dimerize, emitting red fluorescence.(More details can be found on Implementation - Overview- Our final product)
Reference
Tazuke, Yasuhiko, et al. "A new enzymatic assay method of sulfated bile acids in urine." Japanese Journal of Clinical Chemistry 21.4 (1992): 249-258.
Makishima, Makoto, et al. "Identification of a nuclear receptor for bile acids." Science 284.5418 (1999): 1362-1365.
Tu, Hua, Arthur Y. Okamoto, and Bei Shan. "FXR, a bile acid receptor and biological sensor." Trends in cardiovascular medicine 10.1 (2000): 30-35.
Alford, Spencer C., et al. "A fluorogenic red fluorescent protein heterodimer." Chemistry & biology 19.3 (2012): 353-360.
