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Description Design Demonstrate Hardware Notebook Future

In a 2015 research report, there are about 170 million people suffering from hyperuricemia, which is an abnormally high level of uric acid (UA) in the blood, in Mainland China ^[1]. Because UA is slightly soluble in water, too much UA in blood is easy to crystallize in the patients' joints and causes gout, which causes severe pain and affects the life quality of the patients ^[2].

After our human practices in this summer, we found there were many problems in current clinical methods to monitor the UA concentration and treat with hyperuricemia and gout. We list some problems here: 1. For UA detection, the most common way is to go to the hospital for a blood test, which bases on uricase and peroxidase. The result is relatively accurate; however, the hospital procedure is always time-consuming and expensive. The enzymes need to be well-preserved to provide reliable results. Reading data of the blood test requires expensive and complex instruments as well as professional operators. These drawbacks limit the application of the UA detection method in diverse areas. 2. For treatment of hyperuricemia and gout, drug control is the most common way. The drugs include Colchicine to relieve pain and inflammation, Alloprinol to inhibit uric acid production, and Probenecid and Benzbromarone to promote uric acid excretion. However, the drugs need to be taken for a long time, which may cause damage to other organs and tissues easily and it is also difficult for patients to take medicine regularly. If the patient has too much UA to form urate, surgery is needed. It is not only just a palliative method, but also costly and risky. The diseases are easy to relapse.

Based on the above information, we believe it is necessary to propose new devices to achieve the functions of detection and removal of high concentrations of UA in the patients’ body. This year, QHFZ-China referenced the past work past work ^{[3], [4]}, and designed a detector bacterium and a smart mammalian cell to achieve solving the problems. Design of the UA detector in E. coli cells: We engineered E. coli cells to detect UA concentration with the idea of synthetic biology. First of all, we will introduce the core components in our design. Hypothetical uricase regulator (HucR) from Deinococcus radiodurans (D. radiodurans) is a bacterial transcriptional factor, which belongs to the MarR family. It acts as a homo-dimer and binds to a special dyad-symmetrical operator site named hucO in the absence of UA with high affinity ^[4]. When UA is present, HucR dissociates from hucO, allowing expression of a downstream gene. Therefore, HucR-hucO system can be adapted to develop a uric-acid-responsive regulatory system. However, the cell wall and outer membrane of E. coli cells are barriers which cause the concentration of intracellular UA may be different from the concentration outside the cell. That means the HucR-hucO system inside the cell may not react to accurate UA concentration in environment. To solve this problem, we find a protein named YgfU, which is a proton-gradient dependent, low-affinity (Km = 0.5 mM), and high-capacity (Vmax = 715 nmol·min^-1·mg^-1) transporter for importing UA into cytoplasm in E. coli ^[5]. With the key modules mentioned above, we designed a UA detection system in E. coli (Fig. 1). Pc is a constitutive promoter, Pcp6 promoter, and it promotes the expression of HucR and YgfU. If the concentration of uric acid (UA) in environment is low, HucR will bind to PhucR, which inhibits the expression of downstream reporter, dsRed or sfGFP. When extracellular UA is present, YgfU can transport UA into the cytoplasm, which leads to the dissociation of HucR from PhucR, and induces the fluorescent protein expression.

Figure 1. Working mechanism of the uric acid detection system in E. coli.
Design of the UA cleaner in HeLa cell line: As culturing stem cells needs much time, expenditure and great skills, we used an immortal human cell line to do the experiment as a proof of concept in this project. To eliminate the interference of endogenous human urate transporter 1 (URAT1), we avoided using cells from kidney (for example, 293T cells) or intestines (for example, HCT116 cells). As a result, we chose a common cell line, human HeLa cell, which is a cervical cancer cell line as chassis. Three key components are shown in Fig. 2A. The first one is the sensing device of the smart cell, named mammalian urate-dependent transsilencer (mUTS), which constitutively expresses a protein fused by human KRAB (Krueppel-associated box) domain and a hypothetical uricase regulator HucR. HucR works as a homo-dimer and binds to a special dyad-symmetrical operator site named hucO in the absence of UA with high affinity ^[4]. The KRAB domain enhances the inhibition efficiency when HucR-hucO interaction exists ^[6]. Only when the UA level is high enough, HucR will release from hucO, and the downstream gene, secretary uricase / urate oxidase (smUOX) expresses. To improve the control efficiency of HucR-hucO system, we referred the literature ^[3] and used eight tandem hucO modules (hucO8) in our smart cell. Finally, we introduced URAT1 in the system, which can enhance the permeability of cell membrane to urate and facilitate transmembrane transport efficiency ^[7]. All the sequences of the above parts are humanized. They are cloned to the backbone pcDNA3.1(+) separately, and their expression of them is promoted by PCMV promoter.

Figure 2. Gene circuits designed for Uric acid degradation. (A) Schematic diagram showed the main gene parts. (B) Schematic diagram showed the operating principle of the gene circuits.
With the key parts mentioned above, our cell can respond to the UA concentration and start UA degradation if UA level is high. In our experiments, we tried to use eGFP and smUOX as the downstream gene. The fluorescence protein was used to debug the mUTS's function of gene expression regulation, while smUOX was used to convert sparingly soluble uric acid to more soluble allantoin which can be easily metabolized by the kidneys (Fig. 2B).

_{[1] Liu, R., Han, C., Wu, D., Xia, X., Gu, J., Guan, H., ... & Teng, W. (2015). Prevalence of hyperuricemia and gout in mainland China from 2000 to 2014: a systematic review and meta-analysis. BioMed research international, 2015.
[2] Moran, M. E. (2003). Uric acid stone disease. Front Biosci, 8(8), s1339.
[3] Kemmer, C., Gitzinger, M., Daoud-El Baba, M., Djonov, V., Stelling, J., & Fussenegger, M. (2010). Self-sufficient control of urate homeostasis in mice by a synthetic circuit. Nature biotechnology, 28(4), 355.
[4] Liang, C., Xiong, D., Zhang, Y., Mu, S., & Tang, S. Y. (2015). Development of a novel uric-acid-responsive regulatory system in Escherichia coli. Applied microbiology and biotechnology, 99(5), 2267-2275.
[5] Papakostas, K., & Frillingos, S. (2012). Substrate selectivity of YgfU, a uric acid transporter from Escherichia coli. Journal of Biological Chemistry, 287(19), 15684-15695.
[6] Urrutia, R. (2003). KRAB-containing zinc-finger repressor proteins. Genome biology, 4(10), 231.
[7] Miura, D., Anzai, N., Jutabha, P., Chanluang, S., He, X., Fukutomi, T., & Endou, H. (2011). Human urate transporter 1 (hURAT1) mediates the transport of orotate. The Journal of Physiological Sciences, 61(3), 253-257.}