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Background

Modeling

InterLab

Hardware

Our hardware combines the traditional dialysis method with synthetic biology and forms a new approach to solve refractory gout. Considering its' working mechanism, the function is realized by the engineered bacteria and the safety is ensured by the dialysis like device. Our design philosophy achieves an innovation in clinical treatment and meets the requirement of biosafety and functionality which makes our device be of great applicability. Furthermore, our hardware is of great potential to be improved in two aspects. On one side, the microminiaturization of sensors together with the working characteristic of our hardware that it doesn't have to take large volume make it possible for our hardware to become a wearable device (stoma operation can be avoided); on the other side, the hardware could turn into a treatment platform since the application of engineered bacteria could be engineered to metabolize other components in blood and thus solve other kinds of metabolism diseases. It also shows potential to improve the specificity of immunoadsorption which is used in clinics now.

Goal

Build a simulation device to apply Bacillus subtilis and verify the ability of Bacillus subtilis to reduce simulated blood uric acid concentration in the device.

Brief introduction

Based on the theory of hemodialysis, we built a dialysis-like device utilizing B. subtilis to reduce uric acid in simulated human vein and completed two schemes aiming at human body veins that is probably available. The working condition, the penetrability of bacteria and uric acid, and biosafety of our device was fully evaluated.

Why

In our subject, the main idea of the second part is to directly remove blood uric acid. Although B. subtilis is a gram-negative bacterium and human infection of B. subtilis is rarely reported, it is still infeasible to let the bacteria work inside human body considering the risk of causing body's blood coagulation and immune response, which may also impair its function. As a result, we turned to seek inspiration from current medical therapeutics and learnt a lot from hemodialysis. On the one hand, the semipermeable membrane of the dialyzer can efficiently separate bacteria and blood. On the other hand, the larger contact area and high throughput allow the device to ensure the effective exchange of uric acid. Unlike the purpose of conventional dialysis treating uremia, our ultimate goal is to eliminate the uratoma, and we think it is feasible as long as the blood uric acid concentration remains a low level for some time. So we can choose an available vein as a target and avoid arteriovenous fistula which is requisite to support long term hemodialysis in a normal therapy. In consideration of the difficulty of human or animal experiments with hemodialysis, our dialysis-like device included the simulation of human blood vessel to help us test the ability of bacteria under the real situation.

Design

Our dialysis-like device is composed of four parts, the human body blood vessel simulation part, the simulated blood extracorporeal circulation part, the bacterial circulation part, and the dialyzer part. The blood vessel simulation part includes a peristaltic pump, a blood storage conical flask, and silicone tubes. As its name suggests, it works to simulate the function of human veins. The conical flask is used to store the simulated circulation blood. The peristaltic pump provides power. The silicone tube has the same inner diameter and blood flow volume with the vein we chose. The simulated blood extracorporeal circulation part includes a peristaltic pump, connecting pipes and needles, and this part’s function is to pump simulated blood into the dialyzer at a certain rate and then make it return to the simulated blood vessel. The bacteria circulation part includes a peristaltic pump, a bacteria storage conical flask, and connecting pipes. Its function is to make the bacteria be recycled into the hollow fiber of the dialyzer. The HD-150 dialyzer is provided by Chengdu OCI Medical Devices Co., Ltd. Its internal dialysis fibrous membrane area is 1.5m2, and its surface dialysis microporous aperture is average of 7 ~ 9nm. Although this is a high throughput product, we think it is still suitable for our work. We let the bacteria go through the hollow fiber while blood flow through outside of the hollow fiber in order to achieve the purpose of material exchange. The assembling of the device is shown below in figure 1 and figure 2.

Figure 1 sketch map of our hardware

Figure 2 picture of our hardware

Mechanism

The working principle of our mechanism is as the follows. We use medium to simulate human blood, adding uric acid to let the final concentration equals that of the hyperuricemia. Silicone tubes with smooth internal wall are used to mimic blood vessels. We can modulate the flow velocity and flux of our mimetic blood to be as same as the blood flow rate by mediating the peristaltic pump, and our simulated blood extracorporeal circulation part will lead the liquid flow into the dialyzer. In terms of the bacterial circulation part with bacteria solution, we let it have the same flow rate but adverse direction, gaining the effect of convection to accelerate the interchange of material. Meanwhile, the same flow rate of those two parts can avoid ultrafiltration furthest and guarantee the least change of blood elements. What’s more, as the small substances are in free diffusion and under the convection effect, they will mainly diffuse rely on their concentration gradient. In our device the main exchange refers to the uric acid in blood and the small proteins in the bacteria solution. Circumstance will be more complicated in vivo, like more protein will need to be taken into consideration, but as Bacillus Subtilis is a safe strain, we regard this difference to be acceptive.

When uric acid flows into the bacteria solution, bacteria can utilize it. The circulation of bacteria solution can let the concentration of uric acid in the dialysis device keep low. The stable and relatively high concentration gradient in those two parts guarantee the reaction happen at express speed.

Security

security is always what we concern first, and we are aware that the foundation of achieving function is guaranteeing safety.

1)bacteria lekage experiment

Though the theoretical parameters of our device already showed no possibility of bacteria revealing into mimetic blood and the whole device is enclosed that no bacterium can invade, we still designed bacteria leakage experiment and protein leaking experiment to test the safety of our device. bacteria leakage experiment is used to test whether Bacillus Subtilis can penetrate the fibrous wall.

Result

Figure 3 bacteria leakage experiment result

As shown in figure 3, the sample from the opening for the bacteria circulation grew bacteria, but the sample from the opening for the blood circulation was with no bacteria growth, which indicated that the bacteria cannot pass through the hollow fiber.

2)Protein leakage experiment

After ensuring that the bacteria cannot pass through the dialyzer, we considered that bacteria might secret protein when grown or after death, which might pass through the hollow fiber and get into the blood, leading to immune response. So we did protein leakage experiment to find out how much protein can be leaked.

Rusults

Table 1 protein leakage experiment result

The result showed that after the running of the device, protein concentration was the same or even decreased (Table 1). We suspected that it’s due to that the dialyzer can absorb some protein in it (the surface of the fiber tube, eg.), which means that, at least in our experiment, the biosafety of our device is highly promising.

Uric acid permeating experiment

Since we are aiming at using our device on human body, we have to select veins which could be utilized and make simulation of them in our hardware. There are some conditions that we should consider in our selection of veins: blood flow, convenience, clinical usage, safety. At last, we chose cephalic vein and femoral vein.

Cephalic vein(figure 4) is a superficial vein in the arm. It has a diameter ranging from 2.2mm to 5mm^1,2,3 and the blood flow rate of it is around 28ml/min². Cephalic vein is a common site for venipuncture, and it’s also wildly used to construct radio cephalic fistula or brachiocephalic fistula in dialysis2. The extensive clinical usage of it ensures that it could also be used together with our device. But the blood flow rate of it is not so high which means the process of our device’s lowering uric acid concentration in the whole blood would need a long time.

Figure 4 cephalic vein⁹

Femoral vein(figure 5) is a deep vein accompanies the femoral artery in the femoral sheath. It has a average diameter ranging from 4.4mm to 7.9mm^4,5,6, and the blood flow rate of it is around 138ml/min⁴. Venous catheterization can be conducted in femoral vein and femoral vein is also potential for dialysis⁷. Although puncturing into femoral vein is a little more difficult because of the complex distribution of vessels and nerves around it, the flow rate and dialysis use potential ensure its’ work proficiency with our device.

Figure 5 femoral vein¹⁰

The two veins we choose have a same feature that they are not veins with great diameter or flow rate. This is because we want to reduce the harm of immunogen leakage from our device. Most immunogen can be obstructed by the dialyzer, but there is risk that some micromolecular immunogen getting through the hollow fiber membrane and cause immunoreaction. In large veins with high flow rate, the immunoreaction could be much more acute and even do harm to heart.

Experiments are taken to measure the device’s ability of uric acid filtration with the simulation of the two veins. The diameters of silicone tube we settled are 3mm for simulating cephalic vein and 6.4mm for femoral vein. The flow rate inside them are 28ml/min for simulating cephalic vein and 138ml/min for femoral vein. And LB medium with uric acid concentration of 420μg/L, which is the critical concentration of hyperuricemia, is used to simulate the blood and blank LB medium is used to replace the bacteria solution.

Before the experiment, we thought about building a model to predict the variation tendency of the uric acid concentration in two mediums referring to a classic model⁸. We set up assumptions that uric concentration is uniform in the medium. According to Fick’s law, uric acid crossing a section of membrane of unit area in unit time δt is approximately

(1)

Where k is the proportionality constant, and u(t) is the uric acid concentration of blood simulating medium, v(t) is the uric acid concentration in bacteria solution replacing medium.

The quantity of uric acid getting into blank medium is equal to the quantity reduction of the uric acid in the blood simulating medium in δt, so that we can get the equation

(2)

Where V_B is the volume of the blood simulating medium and S is the membrane area of the dialyzer. And we get

(3)

Let δt → 0 gives

(4)

Considering situations in blank medium, we can get the equation,

(5)

Where V_D is the volume of bacteria solution replacing medium. Add (4) and (5)

(6)

Set z=u-v. Thus

(7)

Where α=S_k/V_B +S_k/V_D. Thus,

(8)

A being an arbitrary constant. Take (8) into (4) and we get

(9)

And integrating gives

(10)

Where B is an arbitrary constant. With the same method, we can get

(11)

Since we have the starting conditions that u=u0=420μg/L at t=0, and v=0 at t=0, we can get

(12)

(13)

Because in our experiment, V_B=V_D=V=500ml, thus

(14)

Take (14) into (13) and (12)

(15)

(16)

Result

For every simulated vein, we took out one experiment. For simulated femoral vein, we set the flow rate in the simulated blood extracorporeal circulation part and the bacterial circulation part to be 60ml/min; for simulated cephalic vein, we set the flow rate in the simulated blood extracorporeal circulation part and the bacterial circulation part to be 14ml/min. The experiment is carried out as protocol and the data we get is shown below in table 2:

Table 2 The original HPLC data of uric acid permeating experiment. Because in the two experiment instruments we used to get the HPLC data is different, there is a big different the two sets of data.

We transferred the data into the concentration of uric acid and plotted them. Data-fitting is done to verify that the data conforms to the model, and the result is shown below:

Figure 6 fitting curve and plots of cephalic vein simulation

Table 3 parameters and results of cephalic vein simulation

Figure 7 fitting curve and plots of femoral vein simulation

Table 4 parameters and results of femoral vein simulation

According to figure 6 and figure 7 and parameters (in table 3 and table 4) got from data-fitting, we can find that for femoral vein simulation, the fitting curve matches the plotted data very well. But the match between experiment data and fitting curve of cephalic vein simulation is not so good. While the values of k in the two results are not the same. The reason for those conditions could be that when the flow rate is low, the assumption of uniform uric acid concentration is not valid. Which can be justified in the future work. The results show that efficiency of uric permeating in femoral vein simulation is much better than that in cephalic vein simulation, because for the former one approximately 30 minutes are needed to reach concentration equilibrium but for the later one the time consumed is about 1 hour. It suggests that femoral vein could be more feasible for our device to work with. But there is also an application for cephalic vein. The traditional dialysis demands abundant dialysate fluid to keep the concentration gradient but our bacteria can metabolize uric acid permeated into the bacterial circulation which means we do not need vast dialysate fluid. This working characteristic together with the microminiaturization of sensors make it possible for our hardware to become a wearable device.

Further improvement

In fact, our device is not usable for cleaning of uric acid. With the development of synthetic biology, it is not hard to speculate that more engineered bacteria that have different metabolizing ability can be produced and they can be applied in our device and deal with some other metabolic diseases. It is promising that our device to be a clinical stage for a lot of metabolic diseases in the futher.

Reference

[1]周苏东, 尹成彬, 张开颜,等. 彩色超声探查头静脉走行指导心脏起搏器安置的研究[J]. 中华医学超声杂志:电子版, 2009, 6(3):78-78.

[2]Albayrak R, Yuksel S, Colbay M, et al. Hemodynamic changes in the cephalic vein of patients with hemodialysis arteriovenous fistula[J]. Journal of Clinical Ultrasound Jcu, 2007, 35(3):133.

[3]Boghosian M, Cassel K, Hammes M, et al. Hemodynamics in the cephalic arch of a brachiocephalic fistula[J]. Medical Engineering & Physics, 2014, 36(7):822-830.

[4] Morrison C A, Schreiber M A, Olsen S B, et al. Femoral venous flow dynamics during intraperitoneal and preperitoneal laparoscopic insufflation[J]. Surgical Endoscopy, 1998, 12(10):1213-1216.

[5] Qiu Y, Chen C, Wang L,.et al. Ultrasonic study of deep-vein diameter and blood spectrum changes in full-term pregnant women [J]. J South Med Univ, 2009, 29(1):23-25.

[6]Abraham P, Leftheriotis G, Desvaux B, et al. Diameter and velocity changes in the femoral vein during thermal stress in humans.[J]. Clin Physiol, 1994, 14(1):15-21.

[7]叶朝阳. 血液透析血管通路的理论与实践[M]. 上海医科大学出版社, 2001.

[8] Burghes D N, Borrie M S. Modelling with differential equations[M]. Halsted [distributor], 1981.

[9] https://en.wikipedia.org/wiki/Cephalic_vein#/media/File:Gray574.png

[10] https://en.wikipedia.org/wiki/Femoral_vein#/media/File:Blausen_0609_LegVeins.png

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+                     <li>
                          <a href="https://2017.igem.org/Team:SCU-WestChina/Team">Team</a>
                      </li>
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      <div class="main-wrapper">
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-             <div class="primary-title">Description</div>
+             <div class="primary-title">Hardware</div>
-             <div class="primary-content">Hyperuricemia and refractory gout are caused by the high urate concentration in the blood. Considering there
+             <div class="primary-content">Our hardware combines the traditional dialysis method with synthetic biology and forms a new approach to solve refractory gout. Considering its' working mechanism, the function is realized by the engineered bacteria and the safety is ensured by the dialysis like device. Our design philosophy achieves an innovation in clinical treatment and meets the requirement of biosafety and functionality which makes our device be of great applicability. Furthermore, our hardware is of great potential to be improved in two aspects. On one side, the microminiaturization of sensors together with the working characteristic of our hardware that it doesn't have to take large volume make it possible for our hardware to become a wearable device (stoma operation can be avoided); on the other side, the hardware could turn into a treatment platform since the application of engineered bacteria could be engineered to metabolize other components in blood and thus solve other kinds of metabolism diseases. It also shows potential to improve the specificity of immunoadsorption which is used in clinics now.
-                are still debates on whether the drug should be used in the asymptomatic hyperuricemia patients and the drug
-                resistance of the refractory gout patients, new approaches are eagerly needed. We constructed the urate metabolic
-                pathway in the probiotic E. coli Nissle 1917 and applied it in the gut to reduce the urate concentration
-                in the blood indirectly. In addition, we built a dialysis-like device combined with modified bacteria to
-                utilize the urate directly in the blood. Our solutions provide a suitable, long-term and non-drug treatment
-                for the asymptomatic hyperuricemia patients and an ultimate treatment for the refractory gout patients.
              </div>
          </div>
          <div class="mid-wrapper">
-            <div class="nav left-nav">
+<div class="title-center">Goal</div>
-                <div class="first-nav">
+            <p>
-                    <a class="first-nav-link nav-link" href="javascript:void(0)">Reducing the Urate in the Gut</a>
+               Build a simulation device to apply Bacillus subtilis and verify the ability of Bacillus subtilis to  reduce simulated blood uric acid concentration in the device.
-                    <div class="second-nav">
+            </p>
-                        <a href="#anchor1-1" class="second-nav-link  nav-link">The Pathway</a>
+            <div class="title-center">Brief introduction</div>
-                        <a href="#anchor1-2" class="second-nav-link  nav-link">Promoters Measurement</a>
+            <p>Based on the theory of hemodialysis, we built a dialysis-like device utilizing B. subtilis to reduce uric acid in simulated human vein and completed two schemes aiming at human body veins that is probably available. The working condition, the penetrability of bacteria and uric acid, and biosafety of our device was fully evaluated. </p>
-                        <a href="#anchor1-3" class="second-nav-link  nav-link">Protein Expression</a>
+            <div class="title-center">Why</div>
-                        <a href="#anchor1-4" class="second-nav-link  nav-link">Seperated Crude Extraction Mix Experiment</a>
+            <p>
-                        <a href="#anchor1-5" class="second-nav-link  nav-link">Crude Extraction for Pathway Experiment</a>
+                In our subject, the main idea of the second part is to directly remove blood uric acid. Although <i>B. subtilis</i> is a gram-negative bacterium and human infection of B. subtilis is rarely reported, it is still infeasible to let the bacteria work inside human body considering the risk of causing body's blood coagulation and immune response, which may also impair its function. As a result, we turned to seek inspiration from current medical therapeutics and learnt a lot from hemodialysis. On the one hand, the semipermeable membrane of the dialyzer can efficiently separate bacteria and blood. On the other hand, the larger contact area and high throughput allow the device to ensure the effective exchange of uric acid. Unlike the purpose of conventional dialysis treating uremia, our ultimate goal is to eliminate the uratoma, and we think it is feasible as long as the blood uric acid concentration remains a low level for some time. So we can choose an available vein as a target and avoid arteriovenous fistula which is requisite to support long term hemodialysis in a normal therapy. In consideration of the difficulty of human or animal experiments with hemodialysis, our dialysis-like device included the simulation of human blood vessel to help us test the ability of bacteria under the real situation.
-                        <a href="#anchor1-6" class="second-nav-link  nav-link">YgfU, the Transporter</a>
+            </p>
-                        <a href="#anchor1-7" class="second-nav-link  nav-link">The Parameters for Urate Consuming</a>
+            <div class="title-center">Design</div>
-                        <a href="#anchor1-8" class="second-nav-link  nav-link">Enzymes in the Supernatant?</a>
+            <p>Our dialysis-like device is composed of four parts, the human body blood vessel simulation part, the simulated blood extracorporeal circulation part, the bacterial circulation part, and the dialyzer part. The blood vessel simulation part includes a peristaltic pump, a blood storage conical flask, and silicone tubes. As its name suggests, it works to simulate the function of human veins. The conical flask is used to store the simulated circulation blood. The peristaltic pump provides power. The silicone tube has the same inner diameter and blood flow volume with the vein we chose. The simulated blood extracorporeal circulation part includes a peristaltic pump, connecting pipes and needles, and this part’s function is to pump simulated blood into the dialyzer at a certain rate and then make it return to the simulated blood vessel. The bacteria circulation part includes a peristaltic pump, a bacteria storage conical flask, and connecting pipes. Its function is to make the bacteria be recycled into the hollow fiber of the dialyzer. The HD-150 dialyzer is provided by Chengdu OCI Medical Devices Co., Ltd. Its internal dialysis fibrous membrane area is 1.5m2, and its surface dialysis microporous aperture is average of 7 ~ 9nm. Although this is a high throughput product, we think it is still suitable for our work. We let the bacteria go through the hollow fiber while blood flow through outside of the hollow fiber in order to achieve the purpose of material exchange. The assembling of the device is shown below in figure 1 and figure 2.</p>
-                        <a href="#anchor1-9" class="second-nav-link  nav-link">Animal Experiment</a>
+            <img src="https://static.igem.org/mediawiki/2017/8/8e/T--SCU-WestChina--Wiki-hardware-fg1.png">
-                        <a href="#anchor1-10" class="second-nav-link  nav-link">Future Work</a>
+            <div class="img-describe">
-                    </div>
+            Figure 1 sketch map of our hardware
-                </div>
+            </div>
-                <div class="first-nav">
+            <img src="https://static.igem.org/mediawiki/2017/c/cb/T--SCU-WestChina--Wiki-hardware-fg2.gif">
-                    <a class="first-nav-link nav-link" href="javascript:void(0)">Improvement of Gifu 2016 Project</a>
+            <div class="img-describe">
-                    <div class="second-nav">
+            Figure 2 picture of our hardware
-                        <a href="#anchor2-1" class="second-nav-link  nav-link">The Improvement</a>
+            </div>
-                    </div>
+            <div class="title-center">Mechanism</div>
-                </div>
+            <p>The working principle of our mechanism is as the follows. We use medium to simulate human blood, adding uric acid to let the final concentration equals that of the hyperuricemia. Silicone tubes with smooth internal wall are used to mimic blood vessels. We can modulate the flow velocity and flux of our mimetic blood to be as same as the blood flow rate by mediating the peristaltic pump, and our simulated blood extracorporeal circulation part will lead the liquid flow into the dialyzer. In terms of the bacterial circulation part with bacteria solution, we let it have the same flow rate but adverse direction, gaining the effect of convection to accelerate the interchange of material. Meanwhile, the same flow rate of those two parts can avoid ultrafiltration furthest and guarantee the least change of blood elements. What’s more, as the small substances are in free diffusion and under the convection effect, they will mainly diffuse rely on their concentration gradient. In our device the main exchange refers to the uric acid in blood and the small proteins in the bacteria solution. Circumstance will be more complicated in vivo, like more protein will need to be taken into consideration, but as Bacillus Subtilis is a safe strain, we regard this difference to be acceptive.  </p>
-                <div class="first-nav">
+            <p>When uric acid flows into the bacteria solution, bacteria can utilize it. The circulation of bacteria solution can let the concentration of uric acid in the dialysis device keep low. The stable and relatively high concentration gradient in those two parts guarantee the reaction happen at express speed. </p>
-                    <a class="first-nav-link nav-link" href="javascript:void(0)">Dialysis-like Device</a>
+            <div class="title-center">Security</div>
-                    <div class="second-nav">
+            <p>security is always what we concern first, and we are aware that the foundation of achieving function is guaranteeing safety. </p>
-                        <a href="#anchor3-1" class="second-nav-link  nav-link">The Device</a>
+            <p><b>1)bacteria lekage experiment</b></p>
-                        <a href="#anchor3-2" class="second-nav-link  nav-link">The Bacteria</a>
+            <p>Though the theoretical parameters of our device already showed no possibility of bacteria revealing into mimetic blood and the whole device is enclosed that no bacterium can invade, we still designed bacteria leakage experiment and protein leaking experiment to test the safety of our device. bacteria leakage experiment is used to test whether Bacillus Subtilis can penetrate the fibrous wall.</p>
-                        <a href="#anchor3-3" class="second-nav-link  nav-link">Biosafety of the Device</a>
+            <p><b>Result</b></p>
-                        <a href="#anchor3-4" class="second-nav-link  nav-link">Function of the Device</a>
+            <img src="https://static.igem.org/mediawiki/2017/5/54/T--SCU-WestChina--Wiki-hardware-fg3.png">
-                        <a href="#anchor3-5" class="second-nav-link  nav-link">Bacteria in the Device</a>
+            <div class="img-describe">
-                    </div>
+            Figure 3 bacteria leakage experiment result
-                </div>
+            </div>
+            <p>As shown in figure 3, the sample from the opening for the bacteria circulation grew bacteria, but the sample from the opening for the blood circulation was with no bacteria growth, which indicated that the bacteria cannot pass through the hollow fiber.</p>
+            <p><b>2)Protein leakage experiment</b></p>
+            <p>After ensuring that the bacteria cannot pass through the dialyzer, we considered that bacteria might secret protein when grown or after death, which might pass through the hollow fiber and get into the blood, leading to immune response. So we did protein leakage experiment to find out how much protein can be leaked.</p>
+            <p><b>Rusults</b></p>
+            <img src="https://static.igem.org/mediawiki/2017/b/b6/T--SCU-WestChina--Wiki-hardware-table1.png">
+            <div class="img-describe">
+            Table 1 protein leakage experiment result
+            </div>
+            <p>The result showed that after the running of the device, protein concentration was the same or even decreased (Table 1). We suspected that it’s due to that the dialyzer can absorb some protein in it (the surface of the fiber tube, eg.), which means that, at least in our experiment, the biosafety of our device is highly promising.</p>
+            <div class="title-center">Uric acid permeating experiment</div>
+            <p>Since we are aiming at using our device on human body, we have to select veins which could be utilized and make simulation of them in our hardware. There are some conditions that we should consider in our selection of veins: blood flow, convenience, clinical usage, safety. At last, we chose cephalic vein and femoral vein.</p>
+            <p>Cephalic vein(figure 4) is a superficial vein in the arm. It has a diameter ranging from 2.2mm to 5mm<sup>1,2,3</sup> and the blood flow rate of it is around 28ml/min<sup>2</sup>. Cephalic vein is a common site for venipuncture, and it’s also wildly used to construct radio cephalic fistula or brachiocephalic fistula in dialysis2. The extensive clinical usage of it ensures that it could also be used together with our device. But the blood flow rate of it is not so high which means the process of our device’s lowering uric acid concentration in the whole blood would need a long time. </p>
+            <img src="https://static.igem.org/mediawiki/2017/f/f4/T--SCU-WestChina--Wiki-hardware-fg4.png">
+            <div class="img-describe">
+            Figure 4 cephalic vein<sup>9</sup>
+            </div>
+            <p>Femoral vein(figure 5) is a deep vein accompanies the femoral artery in the femoral sheath. It has a average diameter ranging from 4.4mm to 7.9mm<sup>4,5,6</sup>, and the blood flow rate of it is around 138ml/min<sup>4</sup>. Venous catheterization can be conducted in femoral vein and femoral vein is also potential for dialysis<sup>7</sup>. Although puncturing into femoral vein is a little more difficult because of the complex distribution of vessels and nerves around it, the flow rate and dialysis use potential ensure its’ work proficiency with our device.</p>
+            <img src="https://static.igem.org/mediawiki/2017/6/6a/T--SCU-WestChina--Wiki-hardware-fg5.png">
+            <div class="img-describe">
+            Figure 5 femoral vein<sup>10</sup>
              </div>
-             <div class="right-content">
+            <p>The two veins we choose have a same feature that they are not veins with great diameter or flow rate. This is because we want to reduce the harm of immunogen leakage from our device. Most immunogen can be obstructed by the dialyzer, but there is risk that some micromolecular immunogen getting through the hollow fiber membrane and cause immunoreaction. In large veins with high flow rate, the immunoreaction could be much more acute and even do harm to heart. </p>
-                <div class="title-center">Reducing the urate concentration in the gut for HUA patients.</div>
+            <p>Experiments are taken to measure the device’s ability of uric acid filtration with the simulation of the two veins. The diameters of silicone tube we settled are 3mm for simulating cephalic vein and 6.4mm for femoral vein. The flow rate inside them are 28ml/min for simulating cephalic vein and 138ml/min for femoral vein. And LB medium with uric acid concentration of 420μg/L, which is the critical concentration of hyperuricemia, is used to simulate the blood and blank LB medium is used to replace the bacteria solution.</p>
+            <p>Before the experiment, we thought about building a model to predict the variation tendency of the uric acid concentration in two mediums referring to a classic model<sup>8</sup>. We set up assumptions that uric concentration is uniform in the medium. According to Fick’s law, uric acid crossing a section of membrane of unit area in unit time δt is approximately </p>
+            <img src="https://static.igem.org/mediawiki/2017/6/69/T--SCU-WestChina--Wiki-hardware-eq1.png"><p>(1)</p>
+            <p>Where k is the proportionality constant, and u(t) is the uric acid concentration of blood simulating medium, v(t) is the uric acid concentration in bacteria solution replacing medium. </p>
+            <p>The quantity of uric acid getting into blank medium is equal to the quantity reduction of the uric acid in the blood simulating medium in δt, so that we can get the equation</p>
+            <img src="https://static.igem.org/mediawiki/2017/6/69/T--SCU-WestChina--Wiki-hardware-eq2.png">
+            <p>(2)</p>
+            <p>Where V<sub>B</sub> is the volume of the blood simulating medium and S is the membrane area of the dialyzer. And we get</p>
+            <img src="https://static.igem.org/mediawiki/2017/2/23/T--SCU-WestChina--Wiki-hardware-eq3.png"><p>(3)</p>
+            <p>Let δt → 0 gives</p>
+            <img src="https://static.igem.org/mediawiki/2017/e/ec/T--SCU-WestChina--Wiki-hardware-eq4.png"><p>(4)</p>
+            Considering situations in blank medium, we can get the equation,
+            <img src="https://static.igem.org/mediawiki/2017/c/c8/T--SCU-WestChina--Wiki-hardware-eq5.png"><p>(5)</p>
+            <p>Where V<sub>D</sub> is the volume of bacteria solution replacing medium. Add (4) and (5)</p>
+            <img src="https://static.igem.org/mediawiki/2017/d/d8/T--SCU-WestChina--Wiki-hardware-eq6.png"><p>(6)</p>
+            <p>Set z=u-v. Thus</p>
+            <img src="https://static.igem.org/mediawiki/2017/9/9b/T--SCU-WestChina--Wiki-hardware-eq7.png"><p>(7)</p>
+            <p>Where α=S<sub>k</sub>/V<sub>B</sub> +S<sub>k</sub>/V<sub>D</sub>. Thus,</p>
+            <img src="https://static.igem.org/mediawiki/2017/6/61/T--SCU-WestChina--Wiki-hardware-eq8.png"><p>(8)</p>
+            <p>A being an arbitrary constant. Take (8) into (4) and we get</p>
+            <img src="https://static.igem.org/mediawiki/2017/6/6b/T--SCU-WestChina--Wiki-hardware-eq9.png"><p>(9)</p>
+            <p>And integrating gives</p>
+            <img src="https://static.igem.org/mediawiki/2017/8/8f/T--SCU-WestChina--Wiki-hardware-eq10.png"><p>(10)</p>
+            <p>Where B is an arbitrary constant. With the same method, we can get</p>
+            <img src="https://static.igem.org/mediawiki/2017/7/77/T--SCU-WestChina--Wiki-hardware-eq11.png"><p>(11)</p>
+            <p>Since we have the starting conditions that u=u0=420μg/L at t=0, and v=0 at t=0, we can get</p>
+            <img src="https://static.igem.org/mediawiki/2017/d/d0/T--SCU-WestChina--Wiki-hardware-eq12.png"><p>(12)</p>
+            <img src="https://static.igem.org/mediawiki/2017/a/a9/T--SCU-WestChina--Wiki-hardware-eq13.png"><p>(13)</p>
+            <p>Because in our experiment, V<sub>B</sub>=V<sub>D</sub>=V=500ml, thus</p>
+            <img src="https://static.igem.org/mediawiki/2017/e/ef/T--SCU-WestChina--Wiki-hardware-eq14.png"><p>(14)</p>
+            <p>Take (14) into (13) and (12)</p>
+            <img src="https://static.igem.org/mediawiki/2017/a/ac/T--SCU-WestChina--Wiki-hardware-eq15.png"><p>(15)</p>
+            <img src="https://static.igem.org/mediawiki/2017/4/43/T--SCU-WestChina--Wiki-hardware-eq16.png"><p>(16)</p>
+            <p><b>Result</b></p>
+            <p>For every simulated vein, we took out one experiment. For simulated femoral vein, we set the flow rate in the simulated blood extracorporeal circulation part and the bacterial circulation part to be 60ml/min; for simulated cephalic vein, we set the flow rate in the simulated blood extracorporeal circulation part and the bacterial circulation part to be 14ml/min. The experiment is carried out as protocol and the data we get is shown below in table 2:</p>
+            <img src="https://static.igem.org/mediawiki/2017/6/67/T--SCU-WestChina--Wiki-hardware-table2.png">
+             <div class="img-describe">
+            Table 2 The original HPLC data of uric acid permeating experiment. Because in the two experiment instruments we used to get the HPLC data is different, there is a big different the two sets of data.
+            </div>
+            <p>We transferred the data into the concentration of uric acid and plotted them. Data-fitting is done to verify that the data conforms to the model, and the result is shown below:</p>
+            <img src="https://static.igem.org/mediawiki/2017/6/65/T--SCU-WestChina--Wiki-hardware-fg6.jpeg">
+            <div class="img-describe">
+            Figure 6 fitting curve and plots of cephalic vein simulation
+            </div>
+            <img src="https://static.igem.org/mediawiki/2017/3/38/T--SCU-WestChina--Wiki-hardware-table3.png">
+            <div class="img-describe">
+            Table 3 parameters and results of cephalic vein simulation
+            </div>
+            <img src="https://static.igem.org/mediawiki/2017/e/e8/T--SCU-WestChina--Wiki-hardware-fg7.jpeg">
+            <div class="img-describe">
+            Figure 7 fitting curve and plots of femoral vein simulation
+            </div>
+            <img src="https://static.igem.org/mediawiki/2017/7/76/T--SCU-WestChina--Wiki-hardware-table4.png">
+            <div class="img-describe">
+            Table 4 parameters and results of femoral vein simulation
+            </div>
+            <p>According to figure 6 and figure 7 and parameters (in table 3 and table 4) got from data-fitting, we can find that for femoral vein simulation, the fitting curve matches the plotted data very well. But the match between experiment data and fitting curve of cephalic vein simulation is not so good. While the values of k in the two results are not the same. The reason for those conditions could be that when the flow rate is low, the assumption of uniform uric acid concentration is not valid. Which can be justified in the future work. The results show that efficiency of uric permeating in femoral vein simulation is much better than that in cephalic vein simulation, because for the former one approximately 30 minutes are needed to reach concentration equilibrium but for the later one the time consumed is about 1 hour. It suggests that femoral vein could be more feasible for our device to work with. But there is also an application for cephalic vein. The traditional dialysis demands abundant dialysate fluid to keep the concentration gradient but our bacteria can metabolize uric acid permeated into the bacterial circulation which means we do not need vast dialysate fluid. This working characteristic together with the microminiaturization of sensors make it possible for our hardware to become a wearable device.</p>
+            <div class="title-center">Further improvement</div>
+            <p>In fact, our device is not usable for cleaning of uric acid. With the development of synthetic biology, it is not hard to speculate that more engineered bacteria that have different metabolizing ability can be produced and they can be applied in our device and deal with some other metabolic diseases. It is promising that our device to be a clinical stage for a lot of metabolic diseases in the futher.</p>
+            <div class="title-center">Reference</div>
+            <p>[1]周苏东, 尹成彬, 张开颜,等. 彩色超声探查头静脉走行指导心脏起搏器安置的研究[J]. 中华医学超声杂志:电子版, 2009, 6(3):78-78.</p>
+            <p>[2]Albayrak R, Yuksel S, Colbay M, et al. Hemodynamic changes in the cephalic vein of patients with hemodialysis arteriovenous fistula[J]. Journal of Clinical Ultrasound Jcu, 2007, 35(3):133.</p>
+            <p>[3]Boghosian M, Cassel K, Hammes M, et al. Hemodynamics in the cephalic arch of a brachiocephalic fistula[J]. Medical Engineering & Physics, 2014, 36(7):822-830.</p>
+            <p>[4] Morrison C A, Schreiber M A, Olsen S B, et al. Femoral venous flow dynamics during intraperitoneal and preperitoneal laparoscopic insufflation[J]. Surgical Endoscopy, 1998, 12(10):1213-1216.</p>
+            <p>[5] Qiu Y, Chen C, Wang L,.et al. Ultrasonic study of deep-vein diameter and blood spectrum changes in full-term pregnant women [J]. J South Med Univ, 2009, 29(1):23-25.</p>
+            <p>[6]Abraham P, Leftheriotis G, Desvaux B, et al. Diameter and velocity changes in the femoral vein during thermal stress in humans.[J]. Clin Physiol, 1994, 14(1):15-21.</p>
+            <p>[7]叶朝阳. 血液透析血管通路的理论与实践[M]. 上海医科大学出版社, 2001.</p>
+            <p>[8] Burghes D N, Borrie M S. Modelling with differential equations[M]. Halsted [distributor], 1981.</p>
+            <p>[9] https://en.wikipedia.org/wiki/Cephalic_vein#/media/File:Gray574.png</p>
+            <p>[10] https://en.wikipedia.org/wiki/Femoral_vein#/media/File:Blausen_0609_LegVeins.png</p>
-                <p id="anchor1-1">
-                    There’s a pathway to metabolite urate to allatonin existing in bacteria and plant. The process is shown in the Figure 1.
-                    Urate is catalyzed into hydroxyisourate by uricase (1.7.3.3), then hydroxyisourate can react spontaneously
-                    and form allantoin. Allantoin is highly soluble in water and can be easily excreted from the body. The
-                    abovementioned new uricase drug is based on this mechanism. There are another two enzymes (3.5.2.17,
-.1.1.97) that can speed up the process from hydroxyisourate to allantoin.
-                </p>
-                <img src="https://static.igem.org/mediawiki/2017/8/8e/T--SCU-WestChina--Wiki-Project-Description-fig1.png" alt="">
-                <div class="img-describe">
-                    Figure 1 The metabolic pathway design of the project. The dotted lines represent the enzymes which can accelerate the spontaneous
-                    reaction processes. Urate oxidase (uricase, EC 1.7.3.4) is an enzyme with copper bonds that catalyzes
-                    the oxidative opening of the purine ring of uric acid to form 5-hydroxyisourate. HIU hydrolase (3.5.2.17)
-                    catalyzes the second reaction of the pathway, hydrolyzing 5-hydroxyisourate (HIU), a product of uricase
-                    in the first step to form 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU). OHCU is catalyzed by
-                    OHCU decarboxylase (4.1.1.97).
-                </div>
-                <p>
-                    YgfU is a high-capacity transporter for uric acid in Escherichia coli, which is homologous to nucleobase transporters of
-                    the ubiquitous family NCS2. We plan to overexpress the protein to achieve greater efficiency of uric
-                    acid utilization.
-                </p>
-                <p>
-                    We found detailed information about these metabolic enzymes and the transporter from the BRENDA database (Table 1), and we
-                    plan to use the constitutive promoter family Anderson Promoter in iGEM database to express them. As for
-                    the non-regulated expression pathway, if we want to promote the speed of the reaction to the maximum,
-                    the relative expression quantity of the enzymes must be considered. The rate-limiting step determines
-                    the whole speed of the pathway. Based on that, the extra protein expression of other enzymes may cause
-                    systematic burden in our bacteria. To design a better pathway, we did the modeling of our urate metabolic
-                    pathway according to the known Km and Kcat parameters published before, so as to determine the proper
-                    promoters upstream of different genes in order to optimize the expression with lowest energy consumed.
-                    We got the best ratio of PucL and 4.1.1.97. However, the detailed parameters of PucM are still unknown
-                    because they are hard to measure according to previous studies. As a result, we decide to try different
-                    promoters on PucM to construct the optimum pathway. (For 3.5.2.17, we used the sequence from soybean
-                    at the first time because its kinetic parameters have been reported. But the protein can’t be expressed
-                    in our constitutive promoter gene expression structure.
-                </p>
-                <img src="https://static.igem.org/mediawiki/2017/c/ce/T--SCU-WestChina--Wiki-description-table1.png">
-                <div class="img-describe" id="anchor1-2">
-                    Table 1 Characteristics of the three enzymes and transporter.
-                </div>
-                <p>
-                    Our quantitative gene expression is based on the Anderson‘s Promoters. Anderson's promoters are the most widely used constitutive
-                    promoters family. Those promoters are well characterized by RFP fluorescence. However, a key problem
-                    of generalizing the results of characterizations is that the dynamics of gene expression are influenced
-                    by the protein coded for. The RFP expression result might not be compatible with our gene expression.
-                    In 2016 iGEM competition, team William_and_Mary tried to use insulator RiboJ to make relative expression
-                    levels similar between different proteins. They have verified this phenomenon as a paper published before.
-                    We adopted the same RiboJ insulator design to measure the 10 promoters from Anderson’s Promoters family
-                    by expressing eGFP to avoid the influence brought by different coding sequences for our pathway construction.
-                </p>
-                <p>
-                    The reason why we start to do the measurement is that we need to calculate to evaluate if our pathway is efficient enough
-                    to reduce the urate in the gut based on the gut urate concentration, to theoretically avoid our pathway
-                    expressing unnecessary extra protein to reduce the cell burden as much as possible to maintain colonization.
-                    In this condition, absolute protein concentration expressed by different promoters should be known. We
-                    have to find out the relationship between eGFP quantitative protein concentration and the promoters.
-                    And that's the first time in iGEM to evaluate the absolute protein expression of a promoter with the
-                    help of insulator to predict the metabolic ability of a pathway. Our measurement provided a prospective
-                    for future teams to evaluate the efficiency of a pathway.
-                </p>
-                <P>
-                    To achieve this, we decide to draw a standard curve of eGFP fluorescence-eGFP absolute protein quantity. We expressed the
-                    eGFP-6x his-tag in pET28a driven by T7 promoter. We purified the protein and drew the standard curve
-                    successfully (See measurement, protocols for purification and measurement experiment details). The final
-                    results are as follows.
-                </P>
-                <img src="https://static.igem.org/mediawiki/2017/5/5b/T--SCU-WestChina--Wiki-description-tablefig2.png" alt="">
-                <div class="img-describe">
-                    Table 2 The relative expression capacity of the Anderson's promoters measured by SCU-WestChina 2017.
-                </div>
-                <img src="https://static.igem.org/mediawiki/2017/0/0d/T--SCU-WestChina--Wiki-Project-Description-fig2.png" alt="">
-                <div class="img-describe">
-                    Figure 2 The relative expression capacity of the Anderson's promoters measured by SCU-WestChina 2017. (Bar table). J00 is
-                    short for J23100, J04 is short for J23104, etc.
-                </div>
-                <p>
-                    Based on the eGFP measurement and modeling, we designed our molecular biology experiment. Considering the urate concentration
-                    in the gut (about 100uM) and E. coli Nissle 1917 expression ability, we chose J23100 for YgfU and pucL
-                    expression, J23113 for 4.1.1.97 expression. (See modeling and measurement for details)
-                </p>
-                <p>
-                    We constructed 16 parts for our pathway evaluation.
-                </p>
-                <img src="https://static.igem.org/mediawiki/2017/9/91/T--SCU-WestChina--Wiki-description-table2.png" alt="">
-                <div class="img-describe" id="anchor1-3">
-                    Table 3 The parts related to urate metabolic pathway submitted.
-                </div>
-                <p>
-                    BBa_K2334001-BBa_K2334004 are generators of the enzymes and transporter we planned to use. The strongest Anderson’s Promoter
-                    J23100 is used in these parts to test if the proteins are expressed. Parts were transformed into E. coli
-                    BL21 for expression. We detected our target protein by SDS-PAGE.
-                </p>
-                <p>
-                    It’s obvious that PucL, PucM, 4.1.1.97 were expressed successfully. However, the membrane protein YgfU wasn’t detected in
-                    the lysate precipitation after centrifugation. As an application-targeting project, we didn’t continue
-                    to verify the expression of the protein by Western Blotting or qPCR detection. We tested its function
-                    by adding this gene directly into the pathway to see if it can help the pathway function, which will
-                    be discussed later.
-                </p>
-                <img src="https://static.igem.org/mediawiki/2017/a/a5/T--SCU-WestChina--Wiki-Project-Description-003.png" alt="">
-                <img src="https://static.igem.org/mediawiki/2017/4/4c/T--SCU-WestChina--Wiki-Project-Description-004.png" alt="">
-                <div class="img-describe" id="anchor1-4">
-                    Figure 3 pucL, pucM, 4.1.1.97 expressed successfully driven by J23100+RiboJ+B0034 gene strucutre. The proteins expressed
-                    are noted in the figure. However, the expression of YgfU was not detected.
-                </div>
-                <p>
-                    To test the function of the metabolic enzymes we expressed (pucL, pucM, 4.1.1.97), we used the crude bacteria extraction
-                    (expressed in E. coli BL21) to test directly. Before we started to react, the total protein quantity
-                    of each sample was made the same. 100ul reaction extraction was added into 900ul PBS with urate (PH=8.0).
-                    HPLC was performed after reaction for 2h and heat for 10min. The components of reaction extraction are
-                    described in the table with brackets. (See protocols for more details). The result shows that, pucM and
-.1.1.97 didn’t help much in the pathway in this experiment, which is hard to explain because it’s not
-                    consistent with the experiment we would describe later (Table 4) . Theoretically, after adding pucM and
-.1.1.97, the reactions can attain equilibrium more quickly and perform better in urate consuming when
-                    measured at the same time.
-                </p>
-                <img src="https://static.igem.org/mediawiki/2017/8/8e/T--SCU-WestChina--Wiki-description-table3.png" alt="">
-                <img src="https://static.igem.org/mediawiki/2017/8/8f/T--SCU-WestChina--Wiki-Project-Description-005.png" alt="">
-                <div class="img-describe" id="anchor1-5">
-                    Table 4 Results of separated crude extraction mix experiment. Only with pucL the urate concentration can decrease. The numbers
-                    refer to the HPLC original peak area.
-                </div>
-                <p>
-                    We still ligated two (pucL + pucM) enzymes system and three enzymes (pucL + pucM + 4.1,1,97) in pathway in one plasmid (transformed
-                    in E. coli BL21) to test the function (Table 5). Promoters for pucM are chosen based on the range of
-                    their expression ability. We wanted to elevate the expression level of pucM as much as possible. So,
-                    J23100, J23106, J23107, J23108, J23117 were chosen. Those pathways with different promoters were submitted
-                    as parts and details are in the following table.
-                </p>
-                <img src="https://static.igem.org/mediawiki/2017/6/63/T--SCU-WestChina--Wiki-description-table4.png" alt="">
-                <div class="img-describe">
-                    Table 5 The pathway constructed. PucM is driven by promoters with different strengths.
-                </div>
-                <P>
-                    We used the crude bacteria extraction to test the whole pathway function directly. Before we started to react, the total
-                    protein quantity of each sample was made the same. 100ul crude extraction was added into 900ul PBS with
-                    urate (PH=8.0). HPLC was performed after reaction for 2h and 100 ℃ heat for 10min.The results show that,
-                    pucM did work (Figure 4, Figure 5). The tendency can be explained as follows: when the promoter is too
-                    strong, it causes excessive consuming of energy in the bacteria, and the expression of the main enzyme
-                    pucL is thus limited; when the promoter is too weak, the reaction can’t attain equilibrium as quickly
-                    as with a stronger promoter. So, when measured at the time, if the equilibrium was not attained, the
-                    performance of the pathway with weaker promoter would be worse. Our modeling result is consistent with
-                    our experiment result here. However, the result of separated crude extraction mix experiment can't be
-                    explained with the same theory, because when the continuous measurement was performed, performances of
-                    different reaction systems with PucL protein were always the same when measured at a time.
-                </P>
-                <img src="https://static.igem.org/mediawiki/2017/f/fb/T--SCU-WestChina--Wiki-Project-Description-006.png" alt="">
-                <div class="img-describe">
-                    Figure 4 The result of crude extraction experiment of BBa_K2334007, BBa_K2334008, BBa_K2334009, BBa_K2334010. The numbers
-                    in the figure refer to HPLC original peak area urate decrease. The result shows that the expression ability
-                    of pucM does influence the performance of urate consuming. J23106 perform best in the group.
-                </div>
-                <img src="https://static.igem.org/mediawiki/2017/e/e4/T--SCU-WestChina--Wiki-Project-Description-007.png" alt="">
-                <div class="img-describe" id="anchor1-6">
-                    Figure 5 The result of crude extraction experiment of BBa_K2334012, BBa_K2334013, BBa_K2334014, BBa_K2334015, BBa_K2334016.
-                    The numbers in the figure refer to HPLC original peak area urate decrease. The result shows that the
-                    expression ability of pucM does influence the performance of urate consuming. J23106 perform best in
-                    the group. We compared the effect of pucL together in this group.
-                </div>
-                <P>
-                    As for YgfU, we have mentioned that we couldn’t detect the protein expression via SDS-PAGE. We constructed the part BBa_K2334006,
-                    which consists of K2334004 (J23100 + RiboJ + B0034 + YgfU, Urate Transporter Generator) and K2334001
-                    (J23100 + RiboJ + B0034 + pucL, Urate Oxidase Generator).
-                </P>
-                <P>
-                    We tested the function of YgfU by detecting the urate concentration in the overnight cultured LB medium. In this experiment,
-                    we used pucL(K2334001), pucL + YgfU(K2334004), eGFP(J23100 + RiboJ + B0034 + eGFP, not submitted) in
-                    pSB1C3 vector (transformed E. coli BL21). The result shows that, those constructions can’t reduce the
-                    urate concentration in the LB medium (Figure 6).
-                </P>
-                <img src="https://static.igem.org/mediawiki/2017/3/3e/T--SCU-WestChina--Wiki-Project-Description-008.png" alt="">
-                <div class="img-describe">
-                    Figure 6 pucL, YgfU + pucL & eGFP were cultivated in urate LB medium overnight (Urate concentration was about 500uM). The
-                    initial OD600 of the LB medium was controlled at 0.02. Urate concentration in the LB medium was detected
-                    by HPLC. The values we showed are the raw peak area data. The figure shows that pucL, YgfU + pucL were
-                    not able to reduce the urate concentration in the LB medium.
-                </div>
-                <P>
-                    We doubted that it was because the bacteria cell couldn’t intake urate that our system couldn’t work. To verify it, we measured
-                    the urate concentration in the cell cytoplasm when two different genes [eGFP(J23100 + RiboJ + B0034 +
-                    eGFP, not submitted), pucL(BBa_K2334001)] were expressed in pSB1C3 vector.
-                </P>
-                <P>
-                    To our surprise, the result shows that urate did enter the cytoplasm, but at one point, it’s excluded from the cell and won’t
-                    be taken in again anymore (Figure 7). If uricase exists in the cytoplasm, it can still work to reduce
-                    the urate concentration in the cytoplasm, but even so, urate still can’t be taken in continuously. There’s
-                    a possible unknown mechanism to prevent the urate from entering the cell when the mechanism is activated.
-                    In previous research, YgfU was expressed driven by T7 promoter, which was a furious expression process
-                    that the mechanism couldn’t influence in a short time. When YgfU is constitutively over-expressed, the
-                    mechanism can actually act successfully.
-                </P>
-                <img src="https://static.igem.org/mediawiki/2017/b/b2/T--SCU-WestChina--Wiki-Project-Description-009.png" alt="">
-                <div class="img-describe" id="anchor1-7">
-                    Figure 7 pucL and eGFP were cultivated in urate LB medium overnight (Urate concentration is about 1mM). The initial OD600
-                    of the LB medium was controlled at 0.02. Urate concentration in the LB medium was detected by HPLC. Cytoplasm
-                    urate concentration was detected by HPLC as well. Diluted cell lysate was used for detection.
-                </div>
-                <P>
-                    We tried to figure out the parameters that influence the unknown mechanism. In our experiment, we tried two parameters: 1)
-                    Oxygen. 2) Nitrogen Source.
-                </P>
-                <P>
-                    The reason why we suspected these two parameters is that it’s reported that E. coli can utilize allantoin as nitrogen resource
-                    under anaerobic conditions. Based on that, we designed our experiment to verify if those two parameters
-                    are important for urate utilization. We cultivated eGFP, pucL, YgfU+pucL, LM4 Full, LM4 Optimum (transformed
-                    in E. coli Nissle 1917) in M9/LB medium and aerobic & anaerobic environments (see protocol). Considering
-                    that the number of bacteria can influence the ability of urate consuming, we measured the OD600 of the
-                    bacteria when the sample was ready for HPLC test.
-                </P>
-                <P>
-                    The values in different independent repeated experiments are not always the same. But the tendency is the same in every group:
-                    For urate utilization ability, M9(Anaerobic)＞M9(Aerobic) ＞LB(Anaerobic)＞LB(Aerobic). It means that bad
-                    nutrition environment and anaerobic environment can better reduce the urate level. The urate concentration
-                    in gut is about 100uM-400uM. According to our experiment results, our gene constructions (pucL, YgfU
-                    + pucL, LM4 Full, LM4 Optimum) all have the ability to reduce the urate concentration to the ideal value.
-                    However, we cannot determine which one is the best choice. LM4 Full and LM4 Optimum showed no significant
-                    difference in this urate consuming experiment. Moreover, the LM4 optimum grew better than LM4 Full, which
-                    means that our modeling works well (Table 6).
-                </P>
-                <P>
-                </P>
-                <P>
-                    Furthermore, some LB medium samples, even cultivated in an aerobic condition, showed an decrease in urate concentration.
-                    However, we never found urate concentration reducing in LB medium before, which may be related to the
-                    expression host we chose (BL21 and Nissle 1917).
-                </P>
-                <P>
-                    Meanwhile, We measured the OD600 after cultivation for another 24h, and we found that if the absolute value of the difference
-                    between OD600 at 24h & 48h is below 0.05 (which means that bacteria growth is in the platform phase at
-h), the possibility that the urate concentration decrease is more than 100uM(about 1000 if shown in
-                    HPLC peak area) is 85% on average. We doubted that the decrease of urate was due to the outflow of the
-                    uricase after cell death and lysis. The hypothesis can also interpret why anaerobic & worse nutrition
-                    environment, which is with lower environment capacity, is better for urate reduction.
-                </P>
-                <img src="https://static.igem.org/mediawiki/2017/7/74/T--SCU-WestChina--Wiki-description-table5.png" alt="">
-                <div class="img-describe">
-                    Table 6 Urate consuming parameters test. eGFP refers to gene "J23100 + RiboJ + B0034 + eGFP", pucL refers to part BBa_K2334001,
-                    pucL + YgfU refers to part BBa_K2334006, LM4 Full refers to part BBa_K2334017, and LM4 Optimum refers
-                    to part BBa_K2334013. We use HPLC peak area to present rate consuming directly.
-                </div>
-                <img src="https://static.igem.org/mediawiki/2017/f/fa/T--SCU-WestChina--Wiki-Project-Description-010.png" alt="">
-                <img src="https://static.igem.org/mediawiki/2017/b/bd/T--SCU-WestChina--Wiki-Project-Description-011.png" alt="">
-                <img src="https://static.igem.org/mediawiki/2017/3/3e/T--SCU-WestChina--Wiki-Project-Description-012.png" alt="">
-                <div class="img-describe">
-                    Figure 8 The experiment results of Group 1-3 original HPLC peak area result shown in bar. Green bar is eGFP as control. It's
-                    clear that M9 and anaerobic cultivation performed better for urate consuming.
-                </div>
-                <P>
-                    Here's the original experiment result.
-                </P>
-                <img src="https://static.igem.org/mediawiki/2017/5/5e/T--SCU-WestChina--Wiki-description-tablehide1.png" alt="">
-                <img src="https://static.igem.org/mediawiki/2017/a/ac/T--SCU-WestChina--Wiki-description-tablehide2.png" alt="">
-                <img src="https://static.igem.org/mediawiki/2017/4/4f/T--SCU-WestChina--Wiki-description-tablehide3.png" alt="">
-                    <br></br>
-                <br id="anchor1-8"></br>
-                <p>
-                    To verify the theory whether the consuming of the urate is due to the cell lysis after cell death, we centrifuged the M9
-                    anaerobic cultivated medium and added its 100ul supernatant and 10min-heated 100ul supernatant respectively
-                    into 900ul PBS with 3M Urate(PH=8.0) to react in 37 ℃ for 2h. To our surprise, result shows that there’s
-                    no urate consuming detected for each experiment (Figure 9). We suppose that the enzymes might not be
-                    leaked to the outside, and the bacteria can utilize urate when the environment is fully loaded, which
-                    leaves a pressure on them. More experiment and repeats should be conducted to find the mechanism in the
-                    future.
-                </p>
-                <img src="https://static.igem.org/mediawiki/2017/9/90/T--SCU-WestChina--Wiki-Project-Description-013.png" alt="">
-                <div class="img-describe" id="anchor1-9">
-                    Figure 9 The result shows that the supernatant cannot utilize urate, which is not consistent with the hypothesis we raised
-                    before. It may indicate that the bacteria can utilize the urate in a special stage without leakage.
-                </div>
-                <P>
-                    No matter what, the urate concentration outside is lowered anyway, even in LB medium it can meet our requirement to reduce the urate about 100uM. But the real condition in the human gut is so complicated: a. Nutrition environment is hard to simulate because of the circadian rhythm of food intake, the food eaten and the enzymes released by intestine. b. The effect of gut microbiome. c. the peristalsis process in the intestine. d. the oxygen environment. To evaluate whether our project can work in the real condition, we decided to do the animal experiment.
-                </P>
-                <P>
-                    Our animal experiment is conducted with the permission from the Institutional Review Board of West China Medical Center,
-                    Sichuan University.
-                </P>
-                <P>
-                    Firstly, we measured the ability of bacteria colonization. E .coli Nissle 1917 transformed with eGFP-pSB1C3 was given by
-                    oral gavage. After gavage, we collected the feces every two days and cultivated it in 3x chloramphenicol
-                    LB medium overnight. (See protocols for details). The results showed that, we can still detect Nissle
-left in feces at Day 5 after gavage. However, we can’t detect Nissle 1917 at Day 7. We can conclude
-                    that Nissle 1917 was decolonized or lost its plasmid between Day 5 and Day 7 (Figure 10).
-                </P>
-                <img src="https://static.igem.org/mediawiki/2017/3/30/T--SCU-WestChina--Wiki-Project-Description-014.png" alt="">
-                <img src="https://static.igem.org/mediawiki/2017/d/dc/T--SCU-WestChina--Wiki-Project-Description-015.png" alt="">
-                <img src="https://static.igem.org/mediawiki/2017/7/78/T--SCU-WestChina--Wiki-Project-Description-016.png" alt="">
-                <div class="img-describe" id="anchor1-10">
-                    Figure 9 The result shows that the supernatant cannot utilize urate, which is not consistent with the hypothesis we raised
-                    before. It may indicate that the bacteria can utilize the urate in a special stage without leakage.
-                </div>
-                <div class="title-center">Future Work</div>
-                <P>
+    </div>
-                    To elongate the existence of engineered bacteria in the gut to make a better colonization for human beings and sustain the
+    <script src="https://code.jquery.com/jquery-3.2.1.min.js"></script>
-                    maximum and stable gene expression as much as possible, we decided to use the plasmid to express the
+    <script src="https://maxcdn.bootstrapcdn.com/bootstrap/3.3.7/js/bootstrap.min.js"></script>
-                    gene instead of genome integrated expression to promote the protein production. And we planned to knock
+    <!-- <script src="https://cdn.bootcss.com/echarts/3.7.2/echarts.min.js"></script> -->
-                    out the genes alr, dadX in E. coli Nissle 1917, based on the paper published by In Young Hwang et al.
+</body>
-                    from National Univeristy of Singapore. Alr and dadX are responsible for turning D-type amino acids into
-                    L-type amino acids to form cell walls. The knockout of those two genes leads to the death of the bacteria.
-                    Then we planned to add alr gene into the plasmid backbone, to form a complementary plasmid and to prevent
+</p>
-                    plasmid loss. In the condition, antibiotic resistance marker can be deleted for biosafety, and the selection
+<!--
-                    can be completed by auxotrophic selection.
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-                </P>
+CPU time usage: 0.007 seconds
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-                <div class="img-describe">
+Preprocessor generated node count: 64/1000000
-                    Figure 12 the knockout of genes responsible for D-type amino acids synthesis of E. coli cell wall and formation of a complementary
+Post‐expand include size: 82/2097152 bytes
-                    plasmid, according to National University of Singapore.
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+</div>                  <div class="visualClear"></div>
                  </div>
-                <P>
-                    However, after 2 months of knockout experiment, we failed to knockout the genes. Fortunately, we obtained the strain from
-                    National Univeristy of Singapore after looking for their help and confirmed the character of this strain
-                    of E. coli Nissle 1917 (Δalr, ΔdadX) in our laboratory (Figure 12).
-                </P>
-                <img src="https://static.igem.org/mediawiki/2017/3/36/T--SCU-WestChina--Wiki-Project-Description-019.png" alt="">
-                <div class="img-describe">
-                    Figure 13 The growth ability test with (a) and without (b) D-alanine. Only when the medium is added with D-alanine, the E.
-                    coli Nissle 1917 (Δalr, ΔdadX) can grow.
-                </div>
-                <p>
-                    We’ve successfully ligated alr with the pSB1C3 backbone.
-                </p>
-                <img src="https://static.igem.org/mediawiki/2017/1/10/T--SCU-WestChina--Wiki-Project-Description-020.png" alt="">
-                <div class="img-describe">
-                    Figure 14 alr gene with original promoter and RBS from genome is ligated into pSB1C3 backbone by Gibson Assembly. The map
-                    the plasmid is in the figure.
-                </div>
-                <p>
-                    After several times of attempt, we found it hard to transform the engineered plasmid into this kind of strain in the same
-                    way we applied in E. coli Nissle 1917.
-                </p>
-                <p>
-                    We are going to continue our experiment in the future.
-                </p>
-                <br id="anchor2-1"></br>
-                <div class="title-center">Improvement of Gifu 2016 Project</div>
-                <p>
-                    At the same time, we improved the project of 2016 Gifu iGEM team. We used the same pathway in our project. The difference
-                    is that they used this pathway to clean the bird dropping. Here’s their abstract.
-                </p>
-                <p>
-                    “In Japan, environmental pollution caused by excrement of birds is a problem that should be solved. For example, dieback
-                    of trees and spoiling the cityscape are major problems. Birds’ dropping consists mainly of uric acid
-                    and that is really insoluble. Uric acid can be degraded to soluble material, urea. So, our goal is to
-                    catalyze uric acid to urea and make it possible to wash away dropping by rainwater. We’d like to lead
-                    our project to the solution of the pollution. Considering purine metabolism pathway, three enzymes, urate
-                    oxidase, allantoinase, allantoicase, must be synthesized to degrade uric acid to urea.”
-                </p>
-                <p>
-                    In their project, they intended to reduce the urate concentration outside the cell as well. However, they didn't succeed
-                    in the end due to the lack of the exploration of parameters may influence the result and they only used
-                    uricase in their project. In our project, we constructed the complete pathway and verified the parameters
-                    to reduce the urate concentration outside the cell successfully. For their project, according to our
-                    experiment, we suggested them to use M9 medium to culture the bacteria then spread it to the surface
-                    of the bird dropping.
-                </p>
-                <br id="anchor3-1"></br>
-                <div class="title-center">Dialysis-like device</div>
-                <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. Or even more,
-                </p>
-                <p>
-                    We plan to overexpress the protein regulating the uric acid metabolic pathway in Bacillus subtilis to activate the pathway,
-                    and add an in vitro dialysis device, trying to treat patients with refractory gout who are resistant
-                    to intravenous treatment of uricase drugs.
-                </p>
-                <p>
-                    We designed a dialysis device which injects the needles into the human vein in both directions and allow blood to flow through
-                    it. We used commercialized dialyzer in our design. In this dialyzer, the blood flows through the dialyzer
-                    which only allows small molecules to pass through and the bacteria remains in the another side of the
-                    fiber tube, unable to enter the bloodstream. The blood flow inside the tube is mainly driven by the peristaltic
-                    pump (Figure 14).
-                </p>
-                <img src="https://static.igem.org/mediawiki/2017/9/9f/T--SCU-WestChina--Wiki-Project-Description-021.png" alt="">
-                <div class="img-describe">
-                    Figure 15 The figure of the dialysis-like system.
-                </div>
-                <p>
-                    At the point of blood exchange, blood flows into the dialysis tube; outside the dialysis tube is artificial serum medium.
-                    Small molecules in the blood (such as urate) can be exchanged outside through the dialysis tube.
-                </p>
-                <p id="anchor3-2">
-                    For more details, please visit
-                    <a href="https://2017.igem.org/Team:SCU-WestChina/Hardware">Hardware page</a>.
-                </p>
-                <p>
-                    The strain of the bacteria we use for this device is extremely important due to biosafety reasons. Bacillus subtilis is a
-                    common gram-positive bacterium, which is widely used in engineering field. Human infection of Bacillus
-                    subtilis is rarely reported, so it is not considered as a pathogenic bacterium. Also, in the cause-of-death
-                    statistics of the World Health Organization, no data on B. subtilis infections are present since, even
-                    if reported, they would be “invisible” at the international comparative level due to the coding used
-                    for classification of death causes. In the literature, only a few cases of infections due to B. subtilis
-                    are reported and only one retrospective study describes the isolation of antibiotic-resistant strains
-                    of B. subtilis. Because Bacillus subtilis itself has a uric acid metabolic pathway, we plan to use an
-                    engineered Bacillus subtilis strain which hasn’t been used in the blood related clinic therapy before.
-                </p>
-                <p>
-                    Bacillus subtilis itself contains a urate metabolic pathway. The pucJ/K helps uric acid to be transported into the cell,
-                    and pucL/M converts uric acid into allantoin. pucJKLM can be regulated by PucR protein to initiate the
-                    transcription. Therefore, we decide to overexpress pucR gene in Bacillus subtilis to activate the downstream
-                    uric acid metabolic pathway.
-                </p>
-                <img src="https://static.igem.org/mediawiki/2017/f/f1/T--SCU-WestChina--Wiki-Project-Description-022.png" alt="">
-                <div class="img-describe">
-                    Figure 16 The metabolic pathway for catabolism of purine nucleobases in B. subtilis. The large oval represents the inner
-                    cell membrane of B. subtilis containing transport proteins for purine nucleobases and their catabolites
-                    (black circles) encoded by the given genes.
-                </div>
-                <img src="https://static.igem.org/mediawiki/2017/0/09/T--SCU-WestChina--Wiki-Project-Description-023.png" alt="">
-                <div class="img-describe">
-                    Figure 17 PucR can activate the expression of pucJKLM operon to promote the expression of uric acid metabolism pathway genes.
-                </div>
-                <p>
-                    We chose Bacillus subtilis R179 as our host because it’s used in clinics now to treat gut environment disorder cause by gut
-                    microbiome in China, which has passed through safety evaluation at least in intestine.
-                </p>
-                <p>
-                    We ligated the pucR to Bacillus Subtilis PHT43 plasmid successfully. PucR can be expressed with IPTG induced. However, after
-                    many attempts, we didn’t transform it into Bacillus subtilis R179 successfully before the wiki freeze.
-                    As a result, we didn’t test the pucR function successfully before the wiki freeze.
-                </p>
-                <img src="https://static.igem.org/mediawiki/2017/e/ef/T--SCU-WestChina--Wiki-Project-Description-024.png" alt="">
-                <div class="img-describe">
-                    Figure 18 The plasmid map of pucR-PHT43.
-                </div>
-                <p>
-                    Some Bacillus subtilis strains can induce hemolysis. We tested the hemolysis ability of the Bacillus subtilis strain itself,
-                    Bacillus subtilis WB800 and Bacillus subtilis R179. The result showed that R179 is with little hemolysis
-                    ability. In our experiment, we decided to use R179 as our chassis. Though there was a Bacillus subtilis
-                    strain with no hemolysis ability reported, which can be our future chassis.
-                </p>
-                <img src="https://static.igem.org/mediawiki/2017/1/12/T--SCU-WestChina--Wiki-Project-Description-025.png" alt="">
-                <div class="img-describe">
-                    Figure 19 The hemolysis ability test of Bacillus Subtilis with blood agar plate. The zone of hemolysis of R179 is obviously
-                    smaller than that of WB800, which is widely used in bioengineering field.
-                </div>
-                <p id="anchor3-3">
-                    The details about the hardware can be seen in our
-                    <a href="https://2017.igem.org/Team:SCU-WestChina/Hardware">Hardware page</a>. In description, we’d like to only show the experiment result.
-                </p>
-                <p>
-                    Though the microporous aperture of the fiber tube in the dialyzer is about 7-9nm, we still did the experiment to ensure the
-                    bacteria cannot pass through the membrane. After adding the bacteria culture and sterilized water to
-                    two sides of the fiber tube for more than 12h, we cultivated the liquid sampled from both sides of the
-                    fiber tube. The result showed that no bacteria were detected in the sterilized water side, which indicated
-                    that the membrane can prevent the leakage of the bacteria (Figure 17).
-                </p>
-                <img src="https://static.igem.org/mediawiki/2017/e/ef/T--SCU-WestChina--Wiki-Project-Description-026.png" alt="">
-                <div class="img-describe">
-                    Figure 20 The bacteria leakage experiment. The sample from bacteria culture side grew bacteria. The sample from sterilized
-                    water side is with no bacteria grown, which indicated that the membrane can prevent the leakage of the
-                    bacteria.
-                </div>
-                <p>
-                    After ensuring that the bacteria cannot pass through the dialyzer, we considered that bacteria might secret protein when
-                    grown or after death, which might pass through the fiber tube and get into the blood, leading to the
-                    immune response. So we did the leakage experiment to find out how much protein can be leaked. M9 with
-                    Bacillus subtilis (Bacillus subtilis was grown in LB medium overnight, bacteria was centrifuged and added
-                    into M9 medium) and M9 were added into the bacteria culture flask and simulated blood flask separately.
-                    Samples were taken for BCA protein assay at the time. The device ran for 4 hours to simulate the application
-                    in real condition. After that, samples from bacteria culture flask and stimulated blood flask were taken
-                    for BCA protein assay. To our surprise, the result showed that after the running of the device, protein
-                    concentration was the same or even decreased (Table 7). We suspected that it’s due to that the dialyzer
-                    can absorb some protein in it (the surface of the fiber tube, eg.), which means that, at least in our
-                    experiment, the biosafety of our device is highly promising.
-                </p>
-                <img src="https://static.igem.org/mediawiki/2017/8/84/T--SCU-WestChina--Wiki-description-table7.png" alt="">
-                <div class="img-describe">
-                    Table 7 The experiment of protein leakage experiment.
-                </div>
-                <div class="title-center">Reference</div>
-                <p>
-                    [1] C. Lou, B. Stanton, Y.-J. Chen, B. Munsky, C. A. Voigt, Ribozyme-based insu lator parts buffer synthetic circuits from
-                    genetic context. Nat. Biotechnol. 30, 1137 (2012). doi:10.1038/nbt.2401 pmid:23034349
-                </p>
-                <p>
-                    [2] 2016 William & Mary iGEM Team, https://2016.igem.org/Team:William_and_Mary/RiboJ, 2017/10/01.
-                </p>
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