Difference between revisions of "Team:SDSZ-China/Human Practices"

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                     style='-webkit-transform: translateZ(0);width:160px;margin-left:0'>
  
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                     <li><a href="#Professional">Professional Help</a></li>
+
                     <li><a href="#Description">Project Description</a></li>
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                    <!--                     <ul>
+
                     <ul>
−
                                            <li><a href="#Companies">Contact with Local Pharmaceutical Companies</a></li>
+
                        <li><a href="#Background">Background</a></li>
−
                                            <li><a href="#Scientists">Contact with scientists regarding specific technological issues/
+
                        <li><a href="#Purpose">Purpose</a></li>
−
                                                suggestions</a></li>
+
                        <li><a href="#Overview">Overview of the project</a></li>
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                                        </ul> -->
+
                        <li><a href="#Goal">Our goal</a></li>
−
                    <li><a href="#Outreach">Outreach</a></li>
+
                    </ul>
−
                    <!--                    <ul>
+
                     <li><a href="#Demonstration">Demonstration</a></li>
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                                            <li><a href="#Patients">Contact with Local Hospital and Patients</a></li>
+
 
−
                                            <li><a href="#Centre">Contact with Chengdu Bear Rescue Centre</a></li>
+
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                                        </ul> -->
+
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                     <li><a href="#Survey">Survey</a></li>
+
−
                    <li><a href="#Collaboration">Collaboration</a></li>
+
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                    <li><a href="#Critiria">Critiria</a></li>
+
 
                 </ul>
 
                 </ul>
 
             </div>
 
             </div>
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         <div class="col-sm-10" style="padding-right:5%">
 
         <div class="col-sm-10" style="padding-right:5%">
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             <div class="row">
 
             <div class="row">
−
                 <div class="col-sm-12">
+
                 <h1 id="Description">Project Description</h1>
−
                    <h1 id="Professional">Professional Help</h1>
+
                <h2 id="Background">Background</h2>
−
                    <h2 id="Companies">Contact with Local Pharmaceutical Companies</h2>
+
                <figure class="col-ms-6"><img src='https://static.igem.org/mediawiki/2017/7/70/Pd1.png'
−
                    <figure class="col-sm-6">
+
                        alt='https://static.igem.org/mediawiki/2017/7/70/Pd1.png'/></figure>
−
                        <br>
+
                <p class="col-ms-6">Bear bile, one of the most famous animal drugs in Traditional Chinese Medicine (TCM), has been
−
                        <img src="https://static.igem.org/mediawiki/2017/3/3e/Pharmaceutical_Company.jpeg">
+
                    recorded in ancient Chinese medicine book as a significant method to treat hepatic and biliary
−
                    </figure>
+
                    disorders. UDCA, the effective ingredient of bear bile.Aside from the traditional use of bear bile
−
                    <p>In order to get a clearer picture of UDCA-contained drug’s manufacturing circumstance in China,
+
                    in Chinese medicine, UDCA(ursodeoxycholic acid), the effective ingredient of bear bile acid, has a
−
                        our team visited Tianjin Pacific Chemical & Pharmaceutical Company to interview the experts
+
                    much larger pharmaceutical application. As well as the usage of UDCA in dissolving gallstone, its
−
                        about current chemical producing methods. According to Mr. Xing Ruwen and Mr. Li Jinlong, the
+
                    efficacy in primary biliary cirrhosis and primary sclerosing cholangitis (PSC) as an adjunct to
−
                        most popular method of producing UDCA-contained medicine in the market mainly focuses on
+
                    medical therapy has been well established. Newer indications include its use in the management of
−
                        chemical pharmaceutical procedures. Yet this approach has disadvantages including relatively
+
                    chronic hepatitis, cirrhosis, post liver transplant rejection, graft-versus-host disease and acute
−
                        weak efficacy and potential harm to health of the workers. He praises our aim to produce UDCA
+
                    viral hepatitis, where it not only relieves symptoms of cholestasis but also arrests ongoing
−
                        medic in biopharmaceutical procedure and consider it very promising. Furthermore, he encouraged
+
                    hepatocyte necrosis. However, the increasing demand for bear bile has caused bears to be in an
−
                        us to cut off cost so that our method can be accepted by the market and the patients. This
+
                    endangered state: bear poaching and illegal animal trade have greatly dwindled the number of the
−
                        suggestion helped us to clarify our future goal.</p>
+
                    wild Asiatic black bear. Apart from that, bear bile farming industry in Asia extracts bile through
−
                 </div>
+
                    “milking” from the bears, which is operated through surgically implanting a permanent catheter in
 +
                    the animal&#39;s gallbladder to obtain the drips. It is unquestionable that the bear bile farming
 +
                    process will lead to both physical and psychological damage in bears. </p>
 +
                <p></p>
 +
                <h2 id="Purpose">Purpose</h2>
 +
                <figure class="col-ms-6">&lt;<img src='https://static.igem.org/mediawiki/2017/8/8b/Pd2.png'
 +
                            alt='https://static.igem.org/mediawiki/2017/8/8b/Pd2.png'/></figure>
 +
                <p class="col-ms-6">To find substitute or alternative for bear bile farming, our team will be working on the biological
 +
                    synthesis of the main effective component of this important medicine, UDCA(Ursodeoxycholic Acid).
 +
                    This biological approach will not only be more efficient but also be cheaper than the original
 +
                    chemical approach, which is widely used in the current UDCA synthesis industry. </p>
 +
                <p></p>
 +
                <h2 id="Overview">Overview of the project</h2>
 +
                <p>We found that it is possible to convert the main component of poultry bile, CDCA(Chenodeoxycholic
 +
                    Acid), into UDCA, by employing two enzyme-catalyzed the reactions. First, two enzymes was employed
 +
                    to manage the transformation of CDCA to 7oxo-LCA. In the present of 7a-HSD(7alpha-hydroxysteroid
 +
                    dehydrogenase), CDCA is transformed in to 7oxo-LCA by loosing a pair of hydrogen(2H+ and 2e-), the
 +
                    pair of hydrogen is added to NAD+, the cofactor and the acceptor. The NAD+ is transformed into NADH
 +
                    during the reaction. To regenerate the NAD+ and recycle the reaction, to , the LDH(Lactate
 +
                    dehydrogenase) works on pyruvate and take the pair of hydrogen from NADH and transform the pyruvate
 +
                    to lactate and NADH to NAD+.</p>
 +
                <p>In the second step, the 7oxo-LCA is transformed to UDCA by 7β-HSDH(7beta-hydroxysteroid
 +
                    dehydrogenase)and GDH(glutamate dehydrogenase).The 7β-HSDH works on 7oxo-LCA and take a pair of
 +
                    hydrogen from NADPH(the cofactor for the second step)and add it to 7oxo-LCA and form a beta position
 +
                    7-hydroxyl group, which is our target product UDCA. The GDH works on glucose and take a pair of
 +
                    hydrogen from it and add it to the NADP+, to form NADPH to manage the regeneration of cofactor NADPH
 +
                    for the second step.</p>
 +
                 <p></p>
  
−
                 <br>
+
                 <h2 id="Goal">Our goal</h2>
−
                 <br>
+
                 <figure class="col-ms-6"><img src='https://static.igem.org/mediawiki/2017/2/2e/%E8%83%8C%E6%99%AF5.jpeg'
−
            </div>
+
                        alt='https://static.igem.org/mediawiki/2017/2/2e/%E8%83%8C%E6%99%AF5.jpeg'/></figure>
 +
                <p class="col-ms-6">Our mission is to expression of the four enzymes 7α-HSDH (from ecoli DH5a), 7β-HSDH (from
 +
                    Ruminococcus Torques), GDH (from Bacillus subtilis), and LDH from (Lactobacillus delbruechii subsp.
 +
                    Bulgaricus)and test their activities. By adding the CBD( cellulose binding domain)sequnence to the
 +
                    plasmid we construct, we manage to bind our enzyme on gauze. This specific design excels in 3
 +
                    specific ways: first, by controlling the presence of the gauze in the solution, we can control the
 +
                    process of the reaction. Second, when the target enzyme is bound to cellulose we manage to purify
 +
                    the protein we express. Third, the enzyme binding gauze is employed to a machine including the
 +
                    reaction efficiency measuring system and the enzyme addition controlling system .</p>
 +
                <p></p>
  
−
            <br>
 
−
            <br>
 
−
 
−
            <div class="row">
 
−
                <div class="col-sm-4">
 
−
                    <h3>Mr. Xing Ruwen</h3>
 
−
                    <p>Mr. Xing Ruwen is the chief engineer in Tianjin Pacific Chemical & Pharmaceutical Company. He is
 
−
                        an experienced manager who has been working on Chemical method of producing UDCA-contained drugs
 
−
                        for years.</p>
 
−
                </div>
 
−
                <figure class="col-sm-4">
 
−
                    <br>
 
−
                    <img src="https://static.igem.org/mediawiki/2017/9/94/%E8%8D%AF%E5%8E%82xingruwen.jpeg">
 
−
                </figure>
 
−
                <div class="col-sm-4">
 
−
                </div>
 
−
                <div class="col-sm-12">
 
−
                    <p>Mr. Xing introduces us the traditional chemical procedure to manufacture the UDCA-contained drug.
 
−
                        Also, he leads us to visit the processing workshop and teaches us many valuable knowledge about
 
−
                        drug production. </p>
 
−
                    <p>According to his opinion, chemical method of producing UDCA-contained drug basically has two
 
−
                        disadvantages: the first one is its high risk; the second one is related to profit, a factor
 
−
                        that influences manufacturing the most. For its potential high risk, Mr. Xing conveys that it
 
−
                        comes from the utilization of metal sodium. Sodium is well-known for its highly-reactiveness; it
 
−
                        is able react violently with water, which at the same time release large amount of heat and
 
−
                        might cause potential safety issue to the workers. He praises our thought of avoiding the
 
−
                        dangerousness and very much acknowledges our method. </p>
 
−
                </div>
 
 
             </div>
 
             </div>
  
−
            <br>
 
−
            <br>
 
  
−
             <div class="row">
+
             <h1>Demonstration</h1>
 +
            <h2>Our purpose</h2>
 +
            <p>The purpose of our experiment is to employ 4 enzymes to achieve the transformation of CDCA to UDCA. Our
 +
                experiment is divided into two steps. The first step is to oxidize CDCA into 7oxo-LCA, and the second
 +
                step is to reduce 7oxo-LCA to UDCA。</p>
 +
            <p></p>
 +
            <h2>Adding CBD sequence</h2>
 +
            <p>In order to achieve the purification of the enzymes and automatic control of the start and cease of the
 +
                reaction and the recycling usage of enzyme, we added the sequence of CBD（cellulose binding domain) when
 +
                we construct plasmid and manage to express the enzyme that able to bind with gauze(made of cellulose) ,
 +
                before the reaction, we employed ddH2O to wash over the gauze for 3 times in order to purify the enzymes
 +
                .the gauze is then employed on an enzyme slot.</p>
 +
            <p></p>
 +
            <h2>The two step reaction</h2>
 +
            <h3>The first step</h3>
 +
            <p></p>
 +
            <p><img src='file:////tmp/wps-chentong/ksohtml/wps4XWZob.jpg' alt='img'/></p>
 +
            <h4>Overview</h4>
 +
            <p>In the first reaction 7a-HSDH(7alpha-hydrosteroid dehydrogenase) was employed to transform CDCA into
 +
                7oxo-LCA, with the presents of cofactor NAD+ that works as the acceptor of a pair of hydrogen(2H+and
 +
                2e-) from CDCA, the LDH(lactate dehydrogenase) works on pyruvate that take a pair of hydrogen from NADH
 +
                and add it to the pyruvate to form Lactate and manage to transform the NADH back to NAD+. By doing so,
 +
                we manage the regeneration of cofactor of the first step reaction.</p>
 +
            <h4>Our experiment to testify the function of 2 enzyme of the first step</h4>
 +
            <p>Oxidation of CDCA to 7-oxo-LCA using E. coli 7˛-HSDH and NAD+ regeneration</p>
 +
            <p>CDCA was converted in a 3mL solution containing 150 mM</p>
 +
            <p>phosphate buffer(pH 8.0), 10 mM CDCA, 30 mM sodium pyruvate, 0.2mM NAD+, combined with 3U/ml LDH and1 1
 +
                U/ml E. coli DH5a 7a-HSDH-CBD at room temperature. </p>
 +
            <p>The biotransformation experiments were monitored via ultraviolet spectrophotometer at 340nm and HPLC
 +
                measurements by UV detection at 210 nm, using a mobile phase of methanol–water mixture (final ratio
 +
                80:20,pH 3.5 with phosphoric acid) using C18</p>
 +
            <h4>Result</h4>
 +
            <p>As shown in figure, the transformation was complete after 2.5h. </p>
 +
            <p><img src='file:////tmp/wps-chentong/ksohtml/wpsQrMqMq.jpg' alt='img'/></p>
 +
            <p>The Peak area of 7oxo-LCA sample in different concentration </p>
 +
            <table>
 +
                <thead>
 +
                <tr>
 +
                    <th>The concentration of 7oxo-LCA</th>
 +
                    <th>Peak Area</th>
 +
                </tr>
 +
                </thead>
 +
                <tbody>
 +
                <tr>
 +
                    <td>5mM</td>
 +
                    <td>7565117</td>
 +
                </tr>
 +
                <tr>
 +
                    <td>10mM</td>
 +
                    <td>7569983</td>
 +
                </tr>
 +
                <tr>
 +
                    <td>15mM</td>
 +
                    <td>7574421</td>
 +
                </tr>
 +
                <tr>
 +
                    <td>20mM</td>
 +
                    <td>7580012</td>
 +
                </tr>
 +
                </tbody>
 +
            </table>
 +
            <p></p>
 +
            <p></p>
 +
            <p><img src='file:////tmp/wps-chentong/ksohtml/wpsmFoOfb.jpg' alt='img'/></p>
 +
            <h4>Interpretation</h4>
 +
            <p>According to the HPLC after 150minute, there is no significant increase of 7oxo-LCA, as a result, most of
 +
                the CDCA has been transformed into 7oxo-LCA. And according to the HPLC the final yielding rate is
 +
                94%.</p>
 +
            <p>According to the Absorbance of NADH that shown in figure, the absorbance is decreased significantly after
 +
                150 minute dual to the depleted CDCA that stop the conversion of CDCA to 7oxo-LCA and NADH synthesize.
 +
                Because of the abundant amount of pyruvate in the solution, the LDH that works on pyruvate still
 +
                regenerate the NAD+ by taking a pair of hydrogen from NADH until most of the NADH synthesized
 +
                transformed into NAD+.</p>
 +
            <p></p>
 +
            <p></p>
 +
            <h3>The second step</h3>
 +
            <p><img src='file:////tmp/wps-chentong/ksohtml/wpsMsctJV.jpg' alt='img'/></p>
 +
            <h4>Overview</h4>
 +
            <p>In the second step the 7β-HSDH（7 beta-hydrosteroid dehydrogenase) was employed to transform 7oxo-LCA to
 +
                UDCA. The 7oxo-LCA is transformed to UDCA by adding a pair of hydrogen provide from the cofactor for the
 +
                second step, NADPH. The NADPH loose a pair of hydrogen and form NADP+ and the hydrogen is added to
 +
                7oxo-LCA by 7β-HSDH and form a beta position 7-hydroxyl group which is our target produce UDCA. </p>
 +
            <h4>Our experiment to testify the function of 2 enzymes of the second reaction</h4>
 +
            <p>Reduction of 7-oxo-LCA to UDCA using 7β-HSDH and GDH(NADPH regeneration)</p>
 +
            <p>The 3mL reaction solution containing 150 mM</p>
 +
            <p>phosphate buffer(pH 8.0), 10 mM UDCA, 30 mM glucose, 0.2mM NADP+, combined with 2U/ml 7β-HSDH and 5U/ml
 +
                GDH at room temperature.</p>
 +
            <p>The bioconversion experiment was monitored via HPLC measurements. The sample was analyzed by UV detection
 +
                at 210nm. We testify the synthesize of 7-oxo-LCA and the decrease of UDCA. Using a mobile phase of
 +
                methanol–water mixture (final ratio 80:20,pH 3.5 with phosphoric acid) using C18 .</p>
 +
            <h4>Result</h4>
 +
            <p>As shown in the figure below the reaction was complete at 90min</p>
 +
            <p><img src='file:////tmp/wps-chentong/ksohtml/wpsBcvLdG.png' alt='img'/></p>
 +
            <p></p>
 +
            <table>
 +
                <thead>
 +
                <tr>
 +
                    <th>The concentration of 7oxo-LCA</th>
 +
                    <th>Peak Area</th>
 +
                </tr>
 +
                </thead>
 +
                <tbody>
 +
                <tr>
 +
                    <td>5mM</td>
 +
                    <td>7565117</td>
 +
                </tr>
 +
                <tr>
 +
                    <td>10mM</td>
 +
                    <td>7569983</td>
 +
                </tr>
 +
                <tr>
 +
                    <td>15mM</td>
 +
                    <td>7574421</td>
 +
                </tr>
 +
                <tr>
 +
                    <td>20mM</td>
 +
                    <td>7580012</td>
 +
                </tr>
 +
                </tbody>
 +
            </table>
 +
            <p></p>
 +
            <p><img src='file:////tmp/wps-chentong/ksohtml/wpsHXbzIq.jpg' alt='img'/></p>
 +
            <h4>Interpretation</h4>
 +
            <p>According to the HPLC result after 90minute, there is no significant increase of 7oxo-LCA, as a result,
 +
                most of the UDCA has been transformed into 7oxo-LCA. And according to the HPLC the final yielding rate
 +
                is 93%.</p>
 +
            <p>According to the Absorbance of NADPH that shown in figure, the absorbance is decreased significantly
 +
                after 90 minute dual to the depleted UDCA that stop the conversion of UDCA to 7oxo-LCA and NADP+
 +
                synthesize. Because of the abundant amount of glucose in the solution, the GDH that works on glucose
 +
                still regenerate the NADPH by taking a pair of hydrogen from glucose until most of the NADP+ transformed
 +
                into NADPH.</p>
 +
            <p></p>
 +
            <h2>The implication of the enzyme binding gauze</h2>
 +
            <h3>Description of the purpose of our machine</h3>
 +
            <p>Because of the fact that if the two reactions proceed at the same time, the equilibrium of the reactions
 +
                will shifted towards the reactant, CDCA. Thus because of this, we cannot allow the two reactions to
 +
                proceed at the same time and in the same space. To separate the 2 reaction, we design our reaction
 +
                vessel in the following way: After the first reaction is fully completed, the enzyme slot with gauze
 +
                bond by 7a-HSDH and LDH will be raised up, and in this manner, the first reaction is ceased. After this
 +
                process, we will start the second reaction by dropping the enzyme slot with gauze containing 7β-HSDH and
 +
                GDH.</p>
 +
            <p></p>
 +
            <h3>Testification of our own NADH optical detector</h3>
 +
            <p>To testify the function of our NADH optical detector, the change in the concentration of NADH is an
 +
                essential indicator of the course of the reaction, our devices is design to examine the absorbance of
 +
                NADH at 340nm ultraviolet light. By adding NADH in different concentration. We testify the the
 +
                absorbance of ultraviolet ray of NADH under a 3 mL solution.</p>
 +
            <img src='file:////tmp/wps-chentong/ksohtml/wpsKx4Sdb.jpg' alt='img'/>
 +
            <p>Interpretation of the testification:</p>
 +
            <p>A liner function can be demonstrated base on the date we collect of absorbance of NADH in different
 +
                concentration. Their test values are close to the theoretical value of absorbance. Which means our
 +
                devices manage to detect the absorbance of NADH and its concentration.</p>
 +
            <p></p>
 +
            <h3>The relationship between our experiment result and the controlling of our devices </h3>
 +
            <p>Since the change in the concentration of the cofactor NADH is an essential indicator of the course of the
 +
                first step of the reaction, and solutions with different concentration of NADH absorb different
 +
                proportion of the 340nm ultraviolet ray. So in this way we are able to determine the course of the
 +
                reaction just by tracking the change in the solution’s absorbance of 340nm ultraviolet ray. </p>
 +
            <p><img src='file:////tmp/wps-chentong/ksohtml/wps6bMJJV.jpg' alt='img'/></p>
 +
            <p><img src='file:////tmp/wps-chentong/ksohtml/wpsyu4dgG.jpg' alt='img'/></p>
 +
            <h3>Interpretation</h3>
 +
            <p>We manage to find out that when there is a significant decrease shown on the absorbance of NADH , the
 +
                transformation of CDCA reach its end consider no significant increase of 7oxo-LCA shown in the HPLC
 +
                result. So that we can determine the ceasing point of the reaction.As soon as our devices testify the
 +
                significant decrease of absorbance of NADH, we will raise the first enzyme slot from the reaction
 +
                vessel, and thus in this way end the first reaction. The next step is to drop the second enzyme slot and
 +
                start the second reaction.</p>
 +
            <p></p>
 +
            <p></p>
  
−
                <figure class="col-sm-4">
 
−
                    <br>
 
−
                    <img src="https://static.igem.org/mediawiki/2017/3/3a/%E8%8D%AF%E5%8E%82lijinlong.jpeg">
 
−
                </figure>
 
−
                <div class="col-sm-4">
 
−
                    <h3>Mr. Li Jinlong</h3>
 
−
                    <p>Mr. Li Jinlong works as the production manager and he also participating in marketing
 
−
                        department’s work in Tianjin Pacific Chemical & Pharmaceutical Company.</p>
 
−
                </div>
 
−
                <div class="col-sm-4">
 
−
                </div>
 
−
                <div class="col-sm-12">
 
−
                    <p>Mr. Li patiently answers our questions related to marketing. Analyzing its cost, demand and also
 
−
                        profit, Mr. Li helps us to determined our minds on trying to solve the problem step by step. He
 
−
                        informs that currently the successful UDCA synthesis rate is not hight enough and requires
 
−
                        repeating measures to increase its purification ratio. It is very important for us to consider
 
−
                        more about the balance between cost and profits if we want to further develop our method. Now,
 
−
                        the exported UDCA drugs are highly demanded and we need to find ways to help simplify the
 
−
                        production process and to increase the supply of UDCA-contained drugs.</p>
 
−
                </div>
 
−
 
−
            </div>
 
  
 
             <br>
 
             <br>
−
            <br>
 
−
 
−
            <div class="row">
 
−
                <div class="col-sm-12">
 
−
                    <h3>Interview</h3>
 
−
                    <p>Q：We still have some questions regarding to the market. It would be such a pleasure if you can
 
−
                        share some of your ideas or understandings about them.</p>
 
−
                    <p>A : Please ask, I’m very glad to help you.</p>
 
−
                    <p>Q1: Could you please inform us UDCA medicine’s value in market and its efficacy?</p>
 
−
                    <p>A: UDCA is an cholic acid medicine that has choleretic effect to human bodies. It can cure
 
−
                        various dan diseases like gallstone as well as hepatopathy including hepatic adipose
 
−
                        infiltration. UDCA medicine has stronger healing effect than CDCA medicine while having less
 
−
                        side effects in treatment. However, subtracted from bears, UDCA has a relatively high costs. And
 
−
                        using UDCA directly converted by CDCA mainly has two benefits: first of all, it requires a lower
 
−
                        cost because CDCA is subtracted from poultry like chickens, ducks, and geese; also, use CDCA
 
−
                        instead of UDCA can help protect wild animals (bears.) Also, I consulted experts in our
 
−
                        marketing department about it and they revealed to me that this approach has very high
 
−
                        vendibility that medicines manufactured oversea are in short supply.</p>
 
−
                    <p>Q2: What is the basic principle of this drug design/production?</p>
 
−
                    <p>: First we extract CDCA from the bile of chicken, duck and geese. Then we convert CDCA into UDCA
 
−
                        through two steps of redox reaction. These two substance are isomers than differ in their
 
−
                        three-dimensional structure only.</p>
 
−
                    <p>Q3: We found out that there are only few factories manufactures UDCA medicine. Is it because the
 
−
                        producing process’s complexity? Where is the complexity embodied in specific?</p>
 
−
                    <p>A: In fact, the actual process or technology used is not very complex but the key dangerous
 
−
                        factor is the need of sodium, a dangerous, active metal that burns when it meets with water.
 
−
                        Moreover, to consider this issue from other perspectives, UDCA medicine’s production copes wth
 
−
                        the pressure from people’s safety and environment protection concerns. Therefore there are not
 
−
                        too many factories that manufactures UDCA medicine. </p>
 
−
                    <p>Maybe there are only two factories in China that manufactures this active pharmaceutical
 
−
                        ingredient (API) officially; one is in Shenzhen and the other is in Suzhou.</p>
 
−
                    <p>……After biliary drainage, it is very likely that bears will suffer from diseases all over their
 
−
                        body, which is a torture to them. To resolve this problem, researchers and scientists in our
 
−
                        country wish to convert CDCA in order to receive UDCA. Currently, their methods are deeply
 
−
                        restrained by the use of sodium due to its danger and difficulty. If you can convert CDCA to
 
−
                        UDCA successfully using enzymes, you are making a huge contribution. </p>
 
−
                    <p>Also, there are a lot of problems in the production of UDCA in medical industry. For instance, we
 
−
                        need to consider the safety and profits of this drug. Thus, there are still a lot of aspects
 
−
                        need to be considered and experiments need to be done when involved in industrial
 
−
                        production.</p>
 
−
                    <p>Q: Thank you for your suggestions.</p>
 
−
                    <p>Q4: Could you briefly introduce the issue concerning costs and profits?</p>
 
−
                    <p>A: It is complicated because hydrogen needs to be removed. The chemical composition of UDCA needs
 
−
                        to refine repeatedly, which is tricky. For example, a concentration of 70% of UDCA? require
 
−
                        three times of refinement at least in order to reach the standard. Also, the raw materials or
 
−
                        substances need to be recollected and there will be turnovers and other complex factors during
 
−
                        the process. So the profits is limited. And now the biggest issue is that the danger of
 
−
                        production overweights its potential profits.</p>
 
−
 
−
                </div>
 
−
            </div>
 
−
 
 
             <br>
 
             <br>
 
             <br>
 
             <br>
 +
        </div>
  
−
            <div class="row">
 
−
                <div class="col-sm-12">
 
−
                    <h2 id="Scientists">Contact with scientists regarding specific technological issues/
 
−
                        suggestions</h2>
 
−
                </div>
 
−
                <div class="col-sm-6">
 
−
                    <h3>Prof. Xu Jianhe</h3>
 
−
                    <p>Prof. Xu Jianhe is a well-respected professor in Biocatalyis and Bioprocessing State Key
 
−
                        Laboratory of Bioreactor Engineering in East China University of Science and Technology.</p>
 
−
                </div>
 
−
                <figure class="col-sm-6">
 
−
                    <img src="https://static.igem.org/mediawiki/2017/c/c3/XuJianhe.png">
 
−
                </figure>
 
−
 
−
                <div class="col-sm-12">
 
−
                    <p>With many questions regarding to complex bioprocessing and enzyme catalysis, we write to Prof.
 
−
                        Xu, a very famous professor in China and also expert to get further instructions and
 
−
                        professional help. At the beginning of our experiment, we decided to employ a two enzyme in one
 
−
                        pot catalyzed reaction(see figure.1). However, after our initial attempt, we found out that the
 
−
                        reverse reaction was more prevalent than the forward reaction. Thus because of this, the product
 
−
                        was actually CDCA, the initial reactant, instead of UDCA, our expected product. This conundrum
 
−
                        stopped us from reaching any further into the experiment. At this time, Prof. Xu suggested us to
 
−
                        separate the two enzymes into two steps. (see figure 2) After listening to his suggestion, our
 
−
                        team worked together and produced our new experiment plan with a two step reaction instead of
 
−
                        one. In this way, the end product will not be transformed back into CDCA, and the mass
 
−
                        production of USCA proves to be successful. As well as enhancing the biopharmaceutical effect of
 
−
                        UDCA, we also aims to reduce the production cost. When we were designing the experiment, we
 
−
                        included the usage of cofactors: NADH and NADP+. However, the cost of cofactors can be immense
 
−
                        if they are not regenerated. After hearing about our problem, Prof. Xu suggested us finding
 
−
                        enzymes to help with the regeneration of cofactors. With his suggestion in mind, we eventually
 
−
                        employed GDH(Glutamate Dehydrogenase) to regenerate the cofactor NADP+, and LDH(Lactate
 
−
                        Dehydrogenase) to regenerate the cofactor NADH. Carefully answering our questions and giving us
 
−
                        other valuable information and cases about our topic, Prof. Xu used his actions to encourage us
 
−
                        in refining our experiment and to give it a try.</p>
 
−
                </div>
 
−
            </div>
 
−
 
−
            <br>
 
−
            <br>
 
−
 
−
            <div class="row">
 
−
                <div class="col-sm-12">
 
−
                    <h1 id="Outreach">Outreach</h1>
 
−
                    <h2 id="Patients">Contact with Local Hospital and Patients</h2>
 
−
                    <p>In addition, we contact local patients from Xuanwu hospital who suffered from liver cirrhosis to
 
−
                        discuss the curing effects and prices of various UDCA-contained medicines in the market to
 
−
                        determine our direction. Through our interviews, we understood that current UDCA medicines in
 
−
                        the market are unaffordable for patients with average or below average income in long term
 
−
                        treatment. Also, medicine which price can be accepted by the patients has relatively weak curing
 
−
                        effect. In order to solve this problem, we consulted experts from doctors from Xuanwu Hospital.
 
−
                        She reported that UDCA has a large pharmaceutical application. As well as UDCA’s usage in
 
−
                        dissolving gallstone, its efficacy in primary biliary cirrhosis and primary sclerosing
 
−
                        cholangitis (PSC) as an adjunct to medical therapy has been well established. Newer indications
 
−
                        include its use in the management of chronic hepatitis, cirrhosis, post liver transplant
 
−
                        rejection, graft-versus-host disease and acute viral hepatitis, where it not only relieves
 
−
                        symptoms of cholestasis but also arrests ongoing hepatocyte necrosis.</p>
 
−
                    <h3>Patients interview</h3>
 
−
                    <p>Q：Hi, how are you Mrs. Zhou?</p>
 
−
                    <p>A: Good, good. I’m feeling much better now.</p>
 
−
                    <p>Q: If it’s possible, can you briefly explain to us what exactly are you suffering from?</p>
 
−
                    <p>A: Ahh. It’s not a problem. Well, I’ve been diagnosed with liver cirrhosis for several years now.
 
−
                        And I’ve been constantly taking medicine to treat it. It costs a lot of money. Too
 
−
                        expensive!</p>
 
−
                    <p>Q: What is this medicine that you are taking now that is this expensive?</p>
 
−
                    <p>A: I don’t really know the exact name, but you can have a look at it. It’s a medicine produced in
 
−
                        America.</p>
 
−
                    <p>Q: Oh? America? Why aren’t you taking any of the medicine produced locally.</p>
 
−
                    <p>A: That’s a long story to tell. This medicine I’m taking is supposed to be composed of UDCA. From
 
−
                        what the doctors told me, this medicine is designed to treat diseases like what I have here. But
 
−
                        it seems like only the America version works on my. The locally produced version of this
 
−
                        medicine is chemically synthesized so I guess it’s not really a good thing. And I can feel it
 
−
                        too. Because my stomach often hurts from taking the medicine.</p>
 
−
                    <p>Q: Wait… Are you sure of it? Are you sure that your pain comes from taking the chemically
 
−
                        synthesized medicine?</p>
 
−
                    <p>A: Well yes. The pain usually starts an hour or half after taking the medicine. And the doctors
 
−
                        confirmed my theory as well. They say this chemically synthesized version of the medicine would
 
−
                        usually have gastrointestinal discomfort as a side effect.</p>
 
−
                    <p>Q: Well that’s not good! So have you considered any other choices? Or have the doctors
 
−
                        recommended anything else?</p>
 
−
                    <p>A: Yes. And that’s actually why I’m telling you how expansive the medicine is. The chemically
 
−
                        synthesized version of this medicine is not that expensive. Although it cannot be covered by
 
−
                        medical insurance. It’s definitely affordable. The medicine I’m taking now is indeed very good.
 
−
                        It doesn’t have any side effects. I guess the only side effects is that it is too expensive. I
 
−
                        can’t really afford it now. I really want an alternative. But it sees like the only alternative
 
−
                        is bear bile powder.</p>
 
−
                    <p>Q: What do you know about bear bile powder?</p>
 
−
                    <p>A: I know it’s not extremely expensive so I can probably afford it. But I also know how people
 
−
                        acquire bear bile powder. Ugh, I don’t want to cause that much pain to the poor bears. They say
 
−
                        no trading, no killing right? So that’s not really an option for me.</p>
 
−
                    <p>Q: Yeah. It is indeed very brutal. Well, thanks for your time Mrs. Zhou. We really appreciate
 
−
                        your help.</p>
 
−
                    <p>A: No problem! Have a good day.</p>
 
−
                    <p>Q: You too.</p>
 
−
 
−
                </div>
 
−
 
−
                <br>
 
−
                <br>
 
−
            </div>
 
−
 
−
 
−
            <br>
 
−
            <br>
 
−
 
−
            <div class="row">
 
−
                <div class="col-sm-12">
 
−
                    <h2 id="Centre">Contact with Chengdu Bear Rescue Centre</h2>
 
−
                </div>
 
−
                <figure class="col-sm-6">
 
−
                    <br>
 
−
                    <img src="https://static.igem.org/mediawiki/2017/f/fb/%E7%86%8A%E5%9C%BA.jpeg">
 
−
                </figure>
 
−
                <div class="col-sm-6">
 
−
                    <p>To find out more about the bear bile industry and the protective measures against illegal
 
−
                        hunting, our team, SDSZ-China, went all the way down south to Sichuan province, where the
 
−
                        biggest Asiatic Black Bear rescue centre is located. There, after exchanging conversations with
 
−
                        the staff, we found out that Asiatic Black Bear is listed as endangered on the IUCN red list,
 
−
                        and the most efficient way to rescue bears now is buying bile bears from bear farms. (Since bear
 
−
                        farming is a legal activity in China, there’s no way to officially put a halt on this). After
 
−
                        this trip, we went a bit west and visited a real bear farm where bile is siphoned. There, we
 
−
                        learned the heartbreaking fact that people harvest bear bile only to make traditional Chinese
 
−
                        medicine. Yet as most of the modern world would possibly agree, traditional Chinese medicine
 
−
                        hasn’t prove to have a distinct efficacy in treating diseases, rather it serves as a method to
 
−
                        nourish and build up people’s health conditions. Because of the all the above, the need for an
 
−
                        alternative way to produce UDCA struck us as extremely urgent. </p>
 
−
                </div>
 
−
            </div>
 
−
 
−
            <div class="row">
 
−
                <div class="col-sm-12">
 
−
                    <h1 id="Survey">Survey
 
−
                        <small>SDSZ-iGEM Survey</small>
 
−
                    </h1>
 
−
                    <br>
 
−
                    <p>1. Age</p>
 
−
                    <p>请问您的年龄是？</p>
 
−
                    <p></p>
 
−
                    <p>0-10</p>
 
−
                    <p>11-20</p>
 
−
                    <p>21-30</p>
 
−
                    <p>31-40</p>
 
−
                    <p>41-50</p>
 
−
                    <p>51-60</p>
 
−
                    <p>61-70</p>
 
−
                    <br>
 
−
                    <p>2. How much do you know about Bear Bile Extracting Technology? </p>
 
−
                    <p>您有多了解活熊取胆？</p>
 
−
                    <br>
 
−
                    <p>Nothing 没有听说过</p>
 
−
                    <p>A little bit 只是听说过，没有深入了解</p>
 
−
                    <p>I know this but not quite 有一定的了解</p>
 
−
                    <p>I know this quite well 我对此比较了解</p>
 
−
                    <br>
 
−
                    <p>3. Have you ever known anyone who has or had hepatic or biliary disorders? e.g. hepatitis,
 
−
                        fibrosis, and cirrhosis 您是否认识患有或曾患肝胆相关疾病的人？例如：肝炎，肝硬化等</p>
 
−
                    <br>
 
−
                    <p>Yes 有</p>
 
−
                    <p>No 没有</p>
 
−
                    <br>
 
−
                    <p>4. If the previous answer is “yes”, what’s the patient’s general drug treatment?</p>
 
−
                    <p>如果有，您认识的患者一般使用的是哪类药物？</p>
 
−
                    <br>
 
−
                    <p>Bear bile related drugs (UDCA contained)熊胆相关药物 (含UDCA)</p>
 
−
                    <p>Non-bear bile related drugs 非熊胆相关药物</p>
 
−
                    <br>
 
−
                    <p>5. If the previous answer is “Bear bile related drugs”, what’s the patient’s preference on the
 
−
                        type of drugs listed?</p>
 
−
                    <p>如果使用的是熊胆相关药物，请问您认识的患者倾向于购买以下哪种？</p>
 
−
                    <br>
 
−
                    <p>Imported Drug 进口药 </p>
 
−
                    <p>Domestic Drug 国产药</p>
 
−
                    <br>
 
−
                    <p>6. If you don’t know anyone who has or had hepatic or biliary disorders or the person you know
 
−
                        does not use UDCA drugs, then what’s your personal preference on the type of drugs listed?</p>
 
−
                    <p>如果您不认识患有肝胆相关疾病的人或您认识的患者不使用熊胆相关药物，请问您个人倾向于在购买药物时选择以下哪种？</p>
 
−
                    <br>
 
−
                    <p>Imported Drug 进口药</p>
 
−
                    <p>Domestic Drug 国产药</p>
 
−
                    <br>
 
−
                    <p>7. Please fill in the answers according to your personal experience/thoughts</p>
 
−
                    <p>请您按照情况填写</p>
 
−
                    <br>
 
−
                    <p>I chose the imported drug, because </p>
 
−
                    <p>我选择进口药，因为</p>
 
−
                    <p>
 
−
                        <strong>__</strong><strong>__</strong><strong>__</strong><strong>__</strong><strong>__</strong><strong>__</strong><strong>__</strong><strong>__</strong>___
 
−
                    </p>
 
−
                    <p>
 
−
                        <strong>__</strong><strong>__</strong><strong>__</strong><strong>__</strong><strong>__</strong><strong>__</strong><strong>__</strong><strong>__</strong>___
 
−
                    </p>
 
−
                    <br>
 
−
                    <p>I chose the domestic drug, because</p>
 
−
                    <p>我选择国产药，因为</p>
 
−
                    <br>
 
−
                    <p>
 
−
                        <strong>__</strong><strong>__</strong><strong>__</strong><strong>__</strong><strong>__</strong><strong>__</strong><strong>__</strong><strong>__</strong><strong></strong>
 
−
                    </p>
 
−
                    <p>
 
−
                        <strong>__</strong><strong>__</strong><strong>__</strong><strong>__</strong><strong>__</strong><strong>__</strong><strong>__</strong><strong>__</strong><strong></strong>
 
−
                    </p>
 
−
                    <br>
 
−
                    <p>8. Are you comfortable using/consuming products produced from genetically modified organisms?
 
−
                        Check all categories that apply.</p>
 
−
                    <p>您愿意使用基因改造的产品吗？</p>
 
−
                    <br>
 
−
                    <p>Food 食物</p>
 
−
                    <p>Medication 藥物</p>
 
−
                    <p>Other 其他</p>
 
−
                    <p>Not comfortable with using any GMO products 都不愿意</p>
 
−
                    <br>
 
−
                    <p>9. How does the price of treatment affect your choice? To what extent will your choice be
 
−
                        influenced by it?</p>
 
−
                    <p>治疗的价格会在什么程度上影响您的选择？</p>
 
−
                    <br>
 
−
                    <p>Very likely(to affect) 很大程度</p>
 
−
                    <p>Moderate level 中等程度</p>
 
−
                    <p>Not likely 很小程度</p>
 
−
                    <p>Depends (please write down your explanation to the choice)</p>
 
−
                    <p>
 
−
                        <strong>__</strong><strong>__</strong><strong>__</strong><strong>__</strong><strong>__</strong><strong>__</strong><strong>__</strong><strong>__</strong><strong></strong>
 
−
                    </p>
 
−
                    <br>
 
−
                    <p>10. Are you willing to purchase UDCA contained drug that substitutes the conventional Bear bile
 
−
                        extracting technology? </p>
 
−
                    <p>如果有能够替代活熊取胆的药物，您是否愿意购买？</p>
 
−
                    <br>
 
−
                    <p>Yes 愿意</p>
 
−
                    <p>No 不愿意</p>
 
−
 
−
                </div>
 
−
            </div>
 
−
            <br>
 
−
            <br>
 
−
 
−
            <div class="row">
 
−
                <h1 id="Collaboration">Collaboration</h1>
 
−
                <h2>Intro </h2>
 
−
                <p>Starting this summer, we have formed connections and maintained communications with other high school
 
−
                    and
 
−
                    collegiate teams, and we are glad that we could offer our help to some of the teams, and happy to
 
−
                    receive theirs.</p>
 
−
                <h2>RDFZ-China</h2>
 
−
                <div class="col-sm-6">
 
−
                    <p>We provided lab resources to team RDFZ-China for two times. In the first time, we provided Kit
 
−
                        Plate
 
−
                        4 N4
 
−
                        L12 and Kit Plate 3 G6 I6 F22 to their team. In the second time we provided bacteria solution of
 
−
                        PSB
 
−
                        1c3
 
−
                        with designed restriction enzyme cutting site. Since high school teams do not usually have the
 
−
                        financial
 
−
                        support from school, most of the lab materials used by RDFZ are purchased by their team members.
 
−
                        We
 
−
                        are
 
−
                        very happy to share our lab resources and help keep their experiments going and make their
 
−
                        project
 
−
                        come
 
−
                        true.</p>
 
−
                </div>
 
−
                <p></p>
 
−
                <figure class="col-sm-6"><img src='https://static.igem.org/mediawiki/2017/e/e9/SDSZ_RDFZCollaboration.jpeg'
 
−
                                              alt='https://static.igem.org/mediawiki/2017/e/e9/SDSZ_RDFZCollaboration.jpeg'/>
 
−
                </figure>
 
−
                <p></p>
 
−
                <h2>Shipment Coordination</h2>
 
−
                <p>Because of the fact that the person responsible for receiving part in Genescript has recently
 
−
                    resigned
 
−
                    from his position, so there was a period of confusion where all the teams in China didn’t know who
 
−
                    to
 
−
                    send their parts too. One of our team leader Ruohong Wang actively communicated with the Genescript
 
−
                    company and after rounds of negotiation finally got to know the new recipient of all the team’s
 
−
                    parts.
 
−
                    After knowing this piece of information, Ruohong spread the news the all the teams in China and
 
−
                    helped
 
−
                    all of them with this shipping problem. </p>
 
−
 
−
                <figure class="col-sm-4"><img src='https://static.igem.org/mediawiki/2017/d/db/SDSZ_shipping_1.png'
 
−
                                              alt='https://static.igem.org/mediawiki/2017/d/db/SDSZ_shipping_1.png'/>
 
−
                </figure>
 
−
                <figure class="col-sm-4"><img src='https://static.igem.org/mediawiki/2017/4/44/Sdsz_shipping_2.jpeg'
 
−
                                              alt='https://static.igem.org/mediawiki/2017/4/44/Sdsz_shipping_2.jpeg'/>
 
−
                </figure>
 
−
                <figure class="col-sm-4"><img src='https://static.igem.org/mediawiki/2017/3/3d/SDSZ_shipping_3.jpeg'
 
−
                                              alt='https://static.igem.org/mediawiki/2017/3/3d/SDSZ_shipping_3.jpeg'/>
 
−
                </figure>
 
−
            </div>
 
−
 
−
            <div>
 
−
                <h1 id="Critiria">Critiria</h1>
 
−
                <br>
 
−
                <p><a class="btn btn-warning" href="https://static.igem.org/mediawiki/2017/5/51/SDSZ_china_Integrated.pdf">SDSZ-China
 
−
                    Integrated Human Practice</a></p>
 
−
                <p><a class="btn btn-warning" href="https://static.igem.org/mediawiki/2017/f/f6/HP_Silver.pdf">SDSZ-China
 
−
                    Human Practice Silver</a></p>
 
−
                <p><a class="btn btn-warning" href="https://static.igem.org/mediawiki/2017/a/a7/SDSZ-ChinaHPGold.pdf">SDSZ-China
 
−
                    Human Practice Gold</a></p>
 
−
            </div>
 
−
            <br>
 
−
            <br>
 
−
            <br>
 
−
        </div>
 
 
     </div>
 
     </div>
  
 
     <div class="backgroundpic">
 
     <div class="backgroundpic">
−
         <img src="https://static.igem.org/mediawiki/2017/6/62/%E8%83%8C%E6%99%AF1.jpeg" width="100%"
+
         <img src="https://static.igem.org/mediawiki/2017/e/e3/%E8%83%8C%E6%99%AF2jpeg.jpeg" width="100%"
 
             height="100%" id="bg" alt="">
 
             height="100%" id="bg" alt="">
 
     </div>
 
     </div>
−
 
 
</div>
 
</div>
  

Revision as of 02:52, 2 November 2017


Project

Project Description

Background

https://static.igem.org/mediawiki/2017/7/70/Pd1.png

Bear bile, one of the most famous animal drugs in Traditional Chinese Medicine (TCM), has been recorded in ancient Chinese medicine book as a significant method to treat hepatic and biliary disorders. UDCA, the effective ingredient of bear bile.Aside from the traditional use of bear bile in Chinese medicine, UDCA(ursodeoxycholic acid), the effective ingredient of bear bile acid, has a much larger pharmaceutical application. As well as the usage of UDCA in dissolving gallstone, its efficacy in primary biliary cirrhosis and primary sclerosing cholangitis (PSC) as an adjunct to medical therapy has been well established. Newer indications include its use in the management of chronic hepatitis, cirrhosis, post liver transplant rejection, graft-versus-host disease and acute viral hepatitis, where it not only relieves symptoms of cholestasis but also arrests ongoing hepatocyte necrosis. However, the increasing demand for bear bile has caused bears to be in an endangered state: bear poaching and illegal animal trade have greatly dwindled the number of the wild Asiatic black bear. Apart from that, bear bile farming industry in Asia extracts bile through “milking” from the bears, which is operated through surgically implanting a permanent catheter in the animal's gallbladder to obtain the drips. It is unquestionable that the bear bile farming process will lead to both physical and psychological damage in bears.

Purpose

<https://static.igem.org/mediawiki/2017/8/8b/Pd2.png

To find substitute or alternative for bear bile farming, our team will be working on the biological synthesis of the main effective component of this important medicine, UDCA(Ursodeoxycholic Acid). This biological approach will not only be more efficient but also be cheaper than the original chemical approach, which is widely used in the current UDCA synthesis industry.

Overview of the project

We found that it is possible to convert the main component of poultry bile, CDCA(Chenodeoxycholic Acid), into UDCA, by employing two enzyme-catalyzed the reactions. First, two enzymes was employed to manage the transformation of CDCA to 7oxo-LCA. In the present of 7a-HSD(7alpha-hydroxysteroid dehydrogenase), CDCA is transformed in to 7oxo-LCA by loosing a pair of hydrogen(2H+ and 2e-), the pair of hydrogen is added to NAD+, the cofactor and the acceptor. The NAD+ is transformed into NADH during the reaction. To regenerate the NAD+ and recycle the reaction, to , the LDH(Lactate dehydrogenase) works on pyruvate and take the pair of hydrogen from NADH and transform the pyruvate to lactate and NADH to NAD+.

In the second step, the 7oxo-LCA is transformed to UDCA by 7β-HSDH(7beta-hydroxysteroid dehydrogenase)and GDH(glutamate dehydrogenase).The 7β-HSDH works on 7oxo-LCA and take a pair of hydrogen from NADPH(the cofactor for the second step)and add it to 7oxo-LCA and form a beta position 7-hydroxyl group, which is our target product UDCA. The GDH works on glucose and take a pair of hydrogen from it and add it to the NADP+, to form NADPH to manage the regeneration of cofactor NADPH for the second step.

Our goal

https://static.igem.org/mediawiki/2017/2/2e/%E8%83%8C%E6%99%AF5.jpeg

Our mission is to expression of the four enzymes 7α-HSDH (from ecoli DH5a), 7β-HSDH (from Ruminococcus Torques), GDH (from Bacillus subtilis), and LDH from (Lactobacillus delbruechii subsp. Bulgaricus)and test their activities. By adding the CBD( cellulose binding domain)sequnence to the plasmid we construct, we manage to bind our enzyme on gauze. This specific design excels in 3 specific ways: first, by controlling the presence of the gauze in the solution, we can control the process of the reaction. Second, when the target enzyme is bound to cellulose we manage to purify the protein we express. Third, the enzyme binding gauze is employed to a machine including the reaction efficiency measuring system and the enzyme addition controlling system .

Demonstration

Our purpose

The purpose of our experiment is to employ 4 enzymes to achieve the transformation of CDCA to UDCA. Our experiment is divided into two steps. The first step is to oxidize CDCA into 7oxo-LCA, and the second step is to reduce 7oxo-LCA to UDCA。

Adding CBD sequence

In order to achieve the purification of the enzymes and automatic control of the start and cease of the reaction and the recycling usage of enzyme, we added the sequence of CBD（cellulose binding domain) when we construct plasmid and manage to express the enzyme that able to bind with gauze(made of cellulose) , before the reaction, we employed ddH2O to wash over the gauze for 3 times in order to purify the enzymes .the gauze is then employed on an enzyme slot.

The two step reaction

The first step

img

Overview

In the first reaction 7a-HSDH(7alpha-hydrosteroid dehydrogenase) was employed to transform CDCA into 7oxo-LCA, with the presents of cofactor NAD+ that works as the acceptor of a pair of hydrogen(2H+and 2e-) from CDCA, the LDH(lactate dehydrogenase) works on pyruvate that take a pair of hydrogen from NADH and add it to the pyruvate to form Lactate and manage to transform the NADH back to NAD+. By doing so, we manage the regeneration of cofactor of the first step reaction.

Our experiment to testify the function of 2 enzyme of the first step

Oxidation of CDCA to 7-oxo-LCA using E. coli 7˛-HSDH and NAD+ regeneration

CDCA was converted in a 3mL solution containing 150 mM

phosphate buffer(pH 8.0), 10 mM CDCA, 30 mM sodium pyruvate, 0.2mM NAD+, combined with 3U/ml LDH and1 1 U/ml E. coli DH5a 7a-HSDH-CBD at room temperature.

The biotransformation experiments were monitored via ultraviolet spectrophotometer at 340nm and HPLC measurements by UV detection at 210 nm, using a mobile phase of methanol–water mixture (final ratio 80:20,pH 3.5 with phosphoric acid) using C18

Result

As shown in figure, the transformation was complete after 2.5h.

img

The Peak area of 7oxo-LCA sample in different concentration

The concentration of 7oxo-LCA Peak Area
5mM 7565117
10mM 7569983
15mM 7574421
20mM 7580012

img

Interpretation

According to the HPLC after 150minute, there is no significant increase of 7oxo-LCA, as a result, most of the CDCA has been transformed into 7oxo-LCA. And according to the HPLC the final yielding rate is 94%.

According to the Absorbance of NADH that shown in figure, the absorbance is decreased significantly after 150 minute dual to the depleted CDCA that stop the conversion of CDCA to 7oxo-LCA and NADH synthesize. Because of the abundant amount of pyruvate in the solution, the LDH that works on pyruvate still regenerate the NAD+ by taking a pair of hydrogen from NADH until most of the NADH synthesized transformed into NAD+.

The second step

img

Overview

In the second step the 7β-HSDH（7 beta-hydrosteroid dehydrogenase) was employed to transform 7oxo-LCA to UDCA. The 7oxo-LCA is transformed to UDCA by adding a pair of hydrogen provide from the cofactor for the second step, NADPH. The NADPH loose a pair of hydrogen and form NADP+ and the hydrogen is added to 7oxo-LCA by 7β-HSDH and form a beta position 7-hydroxyl group which is our target produce UDCA.

Our experiment to testify the function of 2 enzymes of the second reaction

Reduction of 7-oxo-LCA to UDCA using 7β-HSDH and GDH(NADPH regeneration)

The 3mL reaction solution containing 150 mM

phosphate buffer(pH 8.0), 10 mM UDCA, 30 mM glucose, 0.2mM NADP+, combined with 2U/ml 7β-HSDH and 5U/ml GDH at room temperature.

The bioconversion experiment was monitored via HPLC measurements. The sample was analyzed by UV detection at 210nm. We testify the synthesize of 7-oxo-LCA and the decrease of UDCA. Using a mobile phase of methanol–water mixture (final ratio 80:20,pH 3.5 with phosphoric acid) using C18 .

Result

As shown in the figure below the reaction was complete at 90min

img

The concentration of 7oxo-LCA Peak Area
5mM 7565117
10mM 7569983
15mM 7574421
20mM 7580012

img

Interpretation

According to the HPLC result after 90minute, there is no significant increase of 7oxo-LCA, as a result, most of the UDCA has been transformed into 7oxo-LCA. And according to the HPLC the final yielding rate is 93%.

According to the Absorbance of NADPH that shown in figure, the absorbance is decreased significantly after 90 minute dual to the depleted UDCA that stop the conversion of UDCA to 7oxo-LCA and NADP+ synthesize. Because of the abundant amount of glucose in the solution, the GDH that works on glucose still regenerate the NADPH by taking a pair of hydrogen from glucose until most of the NADP+ transformed into NADPH.

The implication of the enzyme binding gauze

Description of the purpose of our machine

Because of the fact that if the two reactions proceed at the same time, the equilibrium of the reactions will shifted towards the reactant, CDCA. Thus because of this, we cannot allow the two reactions to proceed at the same time and in the same space. To separate the 2 reaction, we design our reaction vessel in the following way: After the first reaction is fully completed, the enzyme slot with gauze bond by 7a-HSDH and LDH will be raised up, and in this manner, the first reaction is ceased. After this process, we will start the second reaction by dropping the enzyme slot with gauze containing 7β-HSDH and GDH.

Testification of our own NADH optical detector

To testify the function of our NADH optical detector, the change in the concentration of NADH is an essential indicator of the course of the reaction, our devices is design to examine the absorbance of NADH at 340nm ultraviolet light. By adding NADH in different concentration. We testify the the absorbance of ultraviolet ray of NADH under a 3 mL solution.

img

Interpretation of the testification:

A liner function can be demonstrated base on the date we collect of absorbance of NADH in different concentration. Their test values are close to the theoretical value of absorbance. Which means our devices manage to detect the absorbance of NADH and its concentration.

The relationship between our experiment result and the controlling of our devices

Since the change in the concentration of the cofactor NADH is an essential indicator of the course of the first step of the reaction, and solutions with different concentration of NADH absorb different proportion of the 340nm ultraviolet ray. So in this way we are able to determine the course of the reaction just by tracking the change in the solution’s absorbance of 340nm ultraviolet ray.

img

img

Interpretation

We manage to find out that when there is a significant decrease shown on the absorbance of NADH , the transformation of CDCA reach its end consider no significant increase of 7oxo-LCA shown in the HPLC result. So that we can determine the ceasing point of the reaction.As soon as our devices testify the significant decrease of absorbance of NADH, we will raise the first enzyme slot from the reaction vessel, and thus in this way end the first reaction. The next step is to drop the second enzyme slot and start the second reaction.