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                    <span dir="auto">Team:SCU-WestChina/Background</span>
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     <div class="main-wrapper">
 
     <div class="main-wrapper">
 
         <div class="primary-wrapper">
 
         <div class="primary-wrapper">
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             <div class="primary-title">Background</div>
+
             <div class="primary-title">Interlab</div>
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             <div class="primary-content">Uric acid is the end product of purine metabolism in human. Increased production or decreased excretion of uric
+
             <div class="primary-content">How close can the numbers be when fluorescence is measured all around the world?
−
                acid can lead to its accumulation in the body, causing hyperuricemia. If sodium urate crystals deposit from
+
−
                super-saturated extracellular fluids to joints, synovium or other tissues and organs, it will cause a syndrome
+
−
                clinically known as "gout". Controversy still exists on whether to use medications on asymptomatic hyperuricemia,
+
−
                refractory gout can be still hard to treat even with the new drug. And those are the reasons why we start
+
−
                our project.
+
 
             </div>
 
             </div>
 
         </div>
 
         </div>
 
         <div class="mid-wrapper">
 
         <div class="mid-wrapper">
 +
            <div class="title-center">Introduction</div>
 
             <p>
 
             <p>
−
                Uric acid is a product of purine nucleotide catabolism in nucleic acid constituent units. Most mammals and poultries are
+
              Over the past three years, iGEM has advanced the frontiers of science with the biggest interlaboratory studies ever done in synthetic biology. The SCU-WestChina iGEM team is honored to participate in the Interlab study this year.
−
                able to produce uricase, which decomposes uric acid into allantoin, then into NH3, CO2 and H2O. However,
+
−
                humans and apes lack the ability to produce uricase, so in humans uric acid is the end product of purine
+
−
                metabolism. For humans, increased production or decreased excretion of uric acid can lead to its accumulation
+
−
                in the body, causing hyperuricemia. Research shows that hyperuricemia is an independent risk factor of arteriosclerosis,
+
−
                hypertension, heart failure and metabolic syndrome[1].
+
 
             </p>
 
             </p>
−
            <img src="https://static.igem.org/mediawiki/2017/6/68/T--SCU-WestChina--Wiki-Background-image002.png" alt="">
 
−
            <div class="img-describe">
 
−
                Figure 1 Purine catabolism. purine bases are converted, first, into xanthine and, then, into urate for excretion[2].
 
−
            </div>
 
 
             <p>
 
             <p>
−
                As the concentration of uric acid in blood increases, if sodium urate crystals deposit from super-saturated extracellular
+
              The aim of the Interlab study is to analyze the measurement data of fluorescence with a detailed protocol, but different lab equipment and environment. In this way, researchers are able to get an access to compare fluorescence data from laboratories all over the world, in a standardized way.
−
                fluids to joints, synovium or other tissues and organs, it will cause a syndrome clinically known as "gout",
+
−
                including features like arthritis, tophi, uric acid kidney stones and gouty nephropathy. At this stage the
+
−
                patient suffer from acute gouty arthritis, having acute pains in the joint from time to time.
+
 
             </p>
 
             </p>
−
             <img src="https://static.igem.org/mediawiki/2017/7/74/T--SCU-WestChina--Wiki-Background-image004.png" alt="">
+
            <div class="title-center">Protocol</div>
 +
            <p>
 +
              <a href="https://static.igem.org/mediawiki/2017/8/85/InterLab_2017_Plate_Reader_Protocol.pdf">InterLab 2017 Plate Reader Protocol</a> was followed. The device utilized in our Interlab experiments was BioTek Cytation™ 3.
 +
            </p>
 +
            <p>
 +
              For this study, following constructs are used:
 +
            </p> 
 +
            <p>
 +
              •Negative Control: I20270
 +
            </p>
 +
            <p>
 +
              •Positive Control: R0040
 +
            </p>
 +
            <p>
 +
              •Test Device 1: J23101.BCD2.E0040.B0015
 +
            </p>
 +
            <p>
 +
              •Test Device 2: J23106.BCD2.E0040.B0015
 +
            </p>
 +
            <p>
 +
              •Test Device 3: J23117.BCD2.E0040.B0015
 +
            </p>
 +
            <p>
 +
              •Test Device 4: J23101+I13504
 +
            </p>
 +
            <p>
 +
              •Test Device 5: J23106+I13504
 +
            </p>       
 +
            <p>
 +
              •Test Device 6: J23117+I13504
 +
            </p> 
 +
            <p>
 +
              All devices and parts were obtained from the iGEM 2017 InterLab Distribution Kit. Devices were made with a pSB1C3 plasmid backbone and transformed into Escherichia coli DH5α.
 +
            </p> 
 +
            <div class="title-center">Data & Result</div>
 +
             <img src="https://static.igem.org/mediawiki/2017/9/98/T--SCU-WestChina--Wiki-Interlab-Fig1.png" alt="">
 
             <div class="img-describe">
 
             <div class="img-describe">
−
                 Figure 2 The illustration of the gout formation. Urate deposits in the joint, which causes inflammation, swelling or even
+
                 Fig. 1 OD600 curve for each replicate, which suggests that most devices had no influence on the replication ability of the cells, while Device 4 and Device 1 showed much lower growth curves.
−
                 the formation of tophi.
+
            </div>
 +
            <img src="https://static.igem.org/mediawiki/2017/9/9f/T--SCU-WestChina--Wiki-Interlab-Fig2.png" alt="">
 +
            <div class="img-describe">
 +
                 Fig. 2 GFP fluorescence data of each device and replicate measured with an excitation of 485 nm and emission of 530 nm
 +
            </div>
 +
            <img src="https://static.igem.org/mediawiki/2017/0/0c/T--SCU-WestChina--Wiki-Interlab-Fig3.png" alt="">
 +
            <div class="img-describe">
 +
                Fig.3 The ratio of fluorescence to growth for each Interlab device.
 
             </div>
 
             </div>
 
             <p>
 
             <p>
−
                 Refractory gout is caused by recurrent episodes of acute gouty arthritis for several years, manifested as chronic, multiple,
+
                 Results from the Plate Reader indicated that the promoter of the Device 4 is strongest, followed by the promoter of the Device 2 and Device 1.
−
                destructive arthritis with pain and tophi formation and / or uric acid kidney stones. The number of such
+
−
                patients grows with the increasing prevalence of gout and the younger onset of the incidence, and these patients
+
−
                have severe illness and low quality of life. 3% of the 3 million gout patients in the United States suffer
+
−
                from refractory gout. Some patients treated by existing medications get poor result or have intolerance,
+
−
                and the treatments are time-consuming and difficult, so how to treat the disease effectively is a major clinical
+
−
                issue faced by physicians.
+
 
             </p>
 
             </p>
 
             <p>
 
             <p>
−
                 The biochemical basis of refractory gout is hyperuricemia, so the key to the treatment is to control the level of serum uric
+
                 The lowest Intensity was detected in Device 3, Device 5 and Device 6.
−
                acid effectively, the target value of which is
+
            </p>
−
                <356μmol / L (<6.0 mg / dl). The daily production of uric acid by a normal adult is 750mg, 80% of which is endogenous, and
+
−
                    20% of which exogenous. The uric acid gets into the metabolic pool of urate, the daily metabolic rate of
+
−
                    which is about 60%.Of the uric acid metabolized, 1/3 is catabolized in the intestine, and 2/3 is excreted
+
−
                    through the kidneys. This process can maintain a stable level of uric acid in the body, and any problem within
+
−
                    the process can lead to hyperuricemia. </p>
+
−
                    <img src="https://static.igem.org/mediawiki/2017/1/1f/T--SCU-WestChina--Wiki-Background-image006.png" alt="">
+
−
                    <div class="img-describe">
+
−
                        Figure 3 Tophaceous gout with multiple tophi on both hands[3].
+
−
                    </div>
+
−
                    <p>
+
−
                        The mechanism of traditional uric acid–lowering agents is mainly to inhibit xanthine oxidase, reducing uric acid production
+
−
                        and to inhibit renal tubular reabsorption of uric acid, promoting uric acid excretion[4].
+
−
                    </p>
+
−
                    <p>
+
−
                        Controversy still exists on whether to use medications on ordinary patients with hyperuricemia: specialists of rheumatic
+
−
                        and immunologic fields state that hyperuricemia without symptoms of gout does not require drug treatment;
+
−
                        however, in the Cardiology field hyperuricemia is considered as an independent risk factor of atherosclerosis
+
−
                        and need to be controlled by medications[5,6]. And for patients with refractory gout, traditional
+
−
                        drug treatment is slow, taking several years to complete, and for some of them the uric acid level
+
−
                        still cannot get low enough for tophi to dissolve. At the same time, for some patients, age factors
+
−
                        and complications like hypertension, renal insufficiency, diabetes and vascular disease have also
+
−
                        limited drug use on them [7].
+
−
                    </p>
+
−
                    <p>
+
−
                        In recent years, Pegloticase, a new type of uric acid–lowering agent, has brought new solutions for patients with refractory
+
−
                        gout and tophi. Pegloticase is a kind of PEG-modified uric acid oxidase which can transform uric
+
−
                        acid into allantoin, which can be easily excreted. It can reduce the level of serum uric acid effectively,
+
−
                        so as to quickly dissolve tophi and treat chronic gout. The PEG modification helps to improve its
+
−
                        antigenicity, and FDA has approved the marketing of the drug (trade name Krystexxa®) in 2010. In
+
−
                        the 2012 American College of Rheumatology Guidelines for Management of Gout, the drugs was recommended
+
−
                        drug for treatment of refractory gout: “Pegloticase is appropriate for patients with severe gout
+
−
                        disease burden and refractoriness to, or intolerance of, conventional and appropriately dosed urate-lowering
+
−
                        therapy”[8]. However, in clinical practice, it is hard to ignore the immune response caused by injections
+
−
                        (Infusion Reactions). Peter E Lipsky et al. found in 2014 that among the 169 patients treated with
+
−
                        the drug, 41% of them had high-titer anti-uricase antibodies, 40% had high-titer anti-PEG antibodies,
+
−
                        and one patient had high-titer anti-uricase antibodies even after treatment with mere placebo[9].
+
−
                        Because of the presence of high-titer antibodies, the efficacy of Pegloticase is low for one-half
+
−
                        of the patients treated, which means that for the small number of patients, refractory gout is incurable[10].
+
−
                        Furthermore, the price of the medication is high, costing around 3,500 USD per month, which also
+
−
                        limits its clinical application. Moreover, the strong uric acid–lowering effect of the drug can cause
+
−
                        very fast dissolve of tophi, leading to acute gout.
+
−
                    </p>
+
−
                    <img src="https://static.igem.org/mediawiki/2017/e/e1/T--SCU-WestChina--Wiki-Background-image008.png" alt="">
+
−
                    <div class="img-describe">Figure 4 The offical website of Pegloticase.</div>
+
−
                    <p>
+
−
                        Based on the reasons above, our project will put forward a new method to control hyperuricemia and treat
+
−
                        refractory gout from the following two aspects:</p>
+
−
                    <p>
+
−
                        1) Control of ordinary hyperuricemia (in the intestinal tract);
+
−
                    </p>
+
−
                    <p>
+
−
                        2) Treatment of refractory gout when Pegloticase treatment is of no avail (with methods of dialysis)
+
−
                    </p>
+
−
                    <p>
+
−
                        1. Control of ordinary hyperuricemia/gout (in the intestinal tract)</p>
+
−
                    <p>
+
−
                        As mentioned above, current drug targets mainly focus on inhibition of uric acid synthesis and inhibition
+
−
                        of uric acid reabsorption protein, with more attention paid to the latter. The intestinal tract itself,
+
−
                        however, as a place for uric acid production, is a potential new target. The mechanism of uric acid
+
−
                        reabsorption in the intestine is not yet fully elucidated. It is found that the purine metabolism
+
−
                        in the intestine may be an important source of uric acid, and the urate transporter—solute carrier
+
−
                        protein 2 family member 9 (SLC2A9) expressed by the intestinal epithelial cells plays an important
+
−
                        role in the transport of uric acid to the blood to maintain the homeostasis[11,12]. An analysis of
+
−
                        intestinal microbes in patients with gout carried out in 2016 showed that the compared to heathy
+
−
                        adults, the intestinal microbes in gout patients produce more uric acid and have fewer enzymes to
+
−
                        catabolize uric acid, resulting in its accumulation in the body[13]. In a study of the effect of
+
−
                        montmorillonite on adsorbing uric acid and lowering blood uric acid levels carried out by Juntao
+
−
                        Li et al., the uric acid concentration in the blood and urinary tract of the mice (administrated
+
−
                        uric acid intragastrically) decreased with the increase of the amount of montmorillonite (Figure
+
−
                        5); for the hyperuricemia model conducted by intraperitoneal injection of uric acid, the mice of
+
−
                        montmorillonite group (administrated intragastrically) had a significantly lower blood uric acid
+
−
                        level than those in the model group. The conclusion is that montmorillonite can adsorb uric acid
+
−
                        significantly and can reduce the serum uric acid level in mice with hyperuricemia[14]. </p>
+
−
                    <img src="https://static.igem.org/mediawiki/2017/2/28/T--SCU-WestChina--Wiki-Background-image010.png" alt="">
+
−
                    <div class="img-describe">Figure 5 Changes of intestinal and serum uric acid concentrations caused by different doses of montmorillonite[14].
+
−
                    </div>
+
−
                    <p>
+
−
                        Furthermore, Ming Li et al. fed mice with hyperuricemia with lactobacilli which utilizes uridine and inosine (important components
+
−
                        in purine metabolism) well, and found their serum uric acid concentration significantly lowered (Figure
+
−
                        6)[15].
+
−
                    </p>
+
−
                    <p>
+
−
                        Based on the information above, we speculated that the reduction of intestinal uric acid concentration can mean a reduced
+
−
                        serum uric acid concentration, thus treating hyperuricemia. Meanwhile, the use of probiotics offers
+
−
                        a perfect solution for the dispute about whether to use medications to treat hyperuricemia: the blood
+
−
                        uric acid level is lowered through proper application of intestinal microbes without the administration
+
−
                        medications.
+
−
                    </p>
+
−
                    <img src="https://static.igem.org/mediawiki/2017/0/0d/T--SCU-WestChina--Wiki-Background-image013.jpg" alt="">
+
−
                    <div class="img-describe"> Figure 6 Changes of serum uric acid concentrations of the mice after being fed with lactobacilli DM9218
+
−
                        which utilizes uridine and inosine (important components in purine metabolism) well[15].
+
−
                    </div>
+
−
                    <p>We hope to construct a metabolic pathway of uric acid in E. coli Nissle 1917 expressing urate permease
+
−
                        (transporter) on its surface. We try to introduce engineered bacteria into the intestine to consume
+
−
                        intestinal uric acid, thus reducing blood uric acid concentration, so as to treat gout.</p>
+
−
                    <p>2. Treatment of refractory gout when Pegloticase treatment is of no avail (with methods like dialysis)</p>
+
−
                    <p>The key to the treatment of refractory gout is to lower the blood uric acid concentration, but direct
+
−
                        protein contact may be blocked immediately by the IgG antibodies (of a long half-life) of the uricase-resistant
+
−
                        patient, so it is necessary to build a relatively independent immunologically privileged sites. Through
+
−
                        the establishment of a dialysis system, we try to solve the problem of refractory gout. At the same
+
−
                        time, if the efficiency of the device is acceptable, it can be used as an inexpensive treatment regimen
+
−
                        to accelerate the dissolution of gout and may be able to change the principles of treatment for chronic
+
−
                        tophi.</p>
+
−
                    <p>References: </p>
+
−
                    <p>[1] Billiet L, Doaty S, Katz J D, et al. Review of Hyperuricemia as New Marker for Metabolic Syndrome[J].
+
−
                        Isrn Rheumatology, 2014, 2014(5-6):852954. </p>
+
−
                    <p>[2] John L. Tymoczko, Jeremy M Berg, Lubert Stryer. Biochemistry: A Short Course (The 3rd Edition), Page
+
−
                        598, 2013</p>
+
−
                    <p>[3] Barajas-Ochoa A, Castaneda-Sanchez J J, Ramos-Remus C. Is gout an easy-to-treat disease? The importance
+
−
                        of health determinants[J]. Reumatología Clínica, 2017.</p>
+
−
                    <p>[4] 刘湘源. 难治性痛风石性痛风的治疗[J]. 中华临床医师杂志:电子版, 2008, 2(6):5-7.</p>
+
−
                    <p>[5] Wakuda H, Uchida S, Ikeda M, et al. Is hyperuricemia a risk factor for arteriosclerosis? Uric Acid
+
−
                        and arteriosclerosis in apolipoprotein e-deficient mice.[J]. Biological & pharmaceutical bulletin,
+
−
                        2014, 37(12):1866-71.</p>
+
−
                    <p>[6] Krishnan E, Pandya B J, Chung L, et al. Hyperuricemia and the risk for subclinical coronary atherosclerosis--data
+
−
                        from a prospective observational cohort study.[J]. Arthritis Research & Therapy, 2011, 13(2):1-8.</p>
+
−
                    <p>[7] Hershfield M S, Ganson N J, Kelly S J, et al. Induced and pre-existing anti-polyethylene glycol antibody
+
−
                        in a trial of every 3-week dosing of pegloticase for refractory gout, including in organ transplant
+
−
                        recipients.[J]. Arthritis Research & Therapy, 2014, 16(2):R63.</p>
+
−
                    <p>[8] Khanna D, Fitzgerald J D, Khanna P P, et al. 2012 American College of Rheumatology guidelines for
+
−
                        management of gout. Part 1: Systematic nonpharmacologic and pharmacologic therapeutic approaches
+
−
                        to hyperuricemia[J]. Arthritis Care Res, 2012, 64(10):1431.</p>
+
−
                    <p>[9] Lipsky P E, Calabrese L H, Kavanaugh A, et al. Pegloticase immunogenicity: the relationship between
+
−
                        efficacy and antibody development in patients treated for refractory chronic gout[J]. Arthritis Research
+
−
                        & Therapy, 2014, 16(2):R60.</p>
+
−
                    <p>[10] Abeles A M. PEG-ing down (and preventing?) the cause of pegloticase failure[J]. Arthritis Research
+
−
                        & Therapy, 2014, 16(3):1-2.</p>
+
−
                    <p>[11] Vitart V, Rudan I, Hayward C, et al. SLC2A9 is a newly identified urate transporter influencing
+
−
                        serum urate concentration, urate excretion and gout[J]. Nature Genetics, 2008, 40(4):437-442.</p>
+
−
                    <p>[12] Ma Z, Long L H, Liu J, et al. Montmorillonite adsorbs uric acid and increases the excretion of uric
+
−
                        acid from the intestinal tract in mice[J]. Journal of Pharmacy & Pharmacology, 2009, 61(11):1499–1504.</p>
+
−
                    <p>[13] Guo Z, Zhang J, Wang Z, et al. Intestinal Microbiota Distinguish Gout Patients from Healthy Humans.[J].
+
−
                        Scientific Reports, 2016, 6:20602.</p>
+
−
                    <p>[14] Ma Z, Long L H, Liu J, et al. Montmorillonite adsorbs uric acid and increases the excretion of uric
+
−
                        acid from the intestinal tract in mice[J]. Journal of Pharmacy & Pharmacology, 2009, 61(11):1499–1504.</p>
+
−
                    <p>[15] Li M, Yang D, Mei L, et al. Screening and Characterization of Purine Nucleoside Degrading Lactic
+
−
                        Acid Bacteria Isolated from Chinese Sauerkraut and Evaluation of the Serum Uric Acid Lowering Effect
+
−
                        in Hyperuricemic Rats[J]. Plos One, 2014, 9(9):e105577-e105577.</p>
+
−
        </div>
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Team:SCU-WestChina/Background

index

Interlab
How close can the numbers be when fluorescence is measured all around the world?
Introduction

Over the past three years, iGEM has advanced the frontiers of science with the biggest interlaboratory studies ever done in synthetic biology. The SCU-WestChina iGEM team is honored to participate in the Interlab study this year.

The aim of the Interlab study is to analyze the measurement data of fluorescence with a detailed protocol, but different lab equipment and environment. In this way, researchers are able to get an access to compare fluorescence data from laboratories all over the world, in a standardized way.

Protocol

InterLab 2017 Plate Reader Protocol was followed. The device utilized in our Interlab experiments was BioTek Cytation™ 3.

For this study, following constructs are used:

•Negative Control: I20270

•Positive Control: R0040

•Test Device 1: J23101.BCD2.E0040.B0015

•Test Device 2: J23106.BCD2.E0040.B0015

•Test Device 3: J23117.BCD2.E0040.B0015

•Test Device 4: J23101+I13504

•Test Device 5: J23106+I13504

•Test Device 6: J23117+I13504

All devices and parts were obtained from the iGEM 2017 InterLab Distribution Kit. Devices were made with a pSB1C3 plasmid backbone and transformed into Escherichia coli DH5α.

Data & Result
Fig. 1 OD600 curve for each replicate, which suggests that most devices had no influence on the replication ability of the cells, while Device 4 and Device 1 showed much lower growth curves.
Fig. 2 GFP fluorescence data of each device and replicate measured with an excitation of 485 nm and emission of 530 nm
Fig.3 The ratio of fluorescence to growth for each Interlab device.

Results from the Plate Reader indicated that the promoter of the Device 4 is strongest, followed by the promoter of the Device 2 and Device 1.

The lowest Intensity was detected in Device 3, Device 5 and Device 6.

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