Difference between revisions of "Team:SDSZ-China/Parts"

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         <div class="col-sm-10" style="padding-right:5%">
 
         <div class="col-sm-10" style="padding-right:5%">
  
−
             <p>分栏：（所有的格式都不用改直接复制粘贴我希望就可以了）</p>
+
             <table id="Table_2" class="pgrouptable addborder tablesorter" style="width: 925px;" cellpadding="0"
−
            <p>parts（表）<br/></p>
+
                  cellspacing="0">
−
            <p>basic parts</p>
+
                 <thead>
−
            <p>-7alpha-HSDH</p>
+
                 <tr>
−
            <p>-7beta-HSDH</p>
+
                    <th colspan="1" style="width: .5em" nowrap="" class="header"></th>
−
            <p>-LDH</p>
+
                    <th colspan="1" style="width: .5em" nowrap="" class="header"></th>
−
            <p>-GDH</p>
+
                    <th style="width: 7em;" class="header">Name</th>
−
            <p>composite parts</p>
+
                    <th colspan="" style="padding-right:7px;" class="header">Type</th>
−
            <p>-ChBD</p>
+
                    <th colspan="" style="padding-right:7px;" class="header">Description</th>
−
            <p>--ChBD--7alpha-HSDH</p>
+
                    <th colspan="" style="padding-right:7px;" class="header">Designer</th>
−
            <p>--ChBD--7beta-HSDH</p>
+
                    <th colspan="" style="padding-right:7px;" class="header">Length</th>
−
            <p>--ChBD-LDH</p>
+
                    <th class="blank_col"></th>
−
            <p>--ChBD-GDH</p>
+
                 </tr>
−
            <p>--ChBD-GFP</p>
+
                 </thead>
−
            <p>-CBD</p>
+
                 <tbody>
−
            <p>--CBD--7alpha-HSDH</p>
+
−
            <p>--CBD--7beta-HSDH</p>
+
−
            <p>--CBD-LDH</p>
+
−
            <p>--CBD-GDH</p>
+
−
            <p>--CBD-GFP</p>
+
−
            <p>contribution</p>
+
−
            <p>-chbd</p>
+
−
            <p>-cbd</p>
+
−
            <p>-7alpha-HSDH</p>
+
−
            <p>7alpha-HSDH (7alpha-hydroxysteroid dehydrogenase) catalyzes the oxidation of hydroxysteroids at C-7
+
−
                position and back, as it converts NAD+ to NADH and back. It catalyzes the oxidation of CDCA into
+
−
                intermediate product 7-oxo-LAC (7-ketolithocholic acid), where the hydroxyl at C-7 position becomes
+
−
                carbonyl.</p>
+
−
            <p></p>
+
−
            <p>Source</p>
+
−
            <p>The 7alpha-HSDH sequence is cloned from the genome of E.coli DH5alpha through PCR amplification, using
+
−
                the primers designed and synthesized based on its sequence.</p>
+
−
            <p>-7beta-HSDH</p>
+
−
            <p>7beta-HSDH (7beta-hydroxysteroid dehydrogenase) catalyzes the reduction of hydroxysteroids at C-7
+
−
                position and back, as it converts NADPH to NDAP+ and back. It catalyzes the reduction of the
+
−
                intermediate product 7-oxo-LAC (7-ketolithocholic acid) into UDCA, the final product, where the carbonyl
+
−
                at C-7 position becomes hydroxyl.</p>
+
−
            <p></p>
+
−
            <p>Source</p>
+
−
            <p>The 7beta-HSDH DNA is originally from the genome of R. torques ATCC 35915, and is artificially
+
−
                synthesized based on the sequence retrieved from the GenBank.</p>
+
−
            <p>-LDH</p>
+
−
            <p>LDH (lactate dehydrogenase) catalyzes the conversion of lactate to pyruvic acid and back, as it converts
+
−
                NAD+ to NADH and back. It is used to catalyze the reduction of pyruvate into lactate, with the presence
+
−
                of NADH, which is oxidized into NAD+ at the same time.</p>
+
−
            <p></p>
+
−
            <p>Source</p>
+
−
            <p>The LDH DNA is originally from the genome of Lactobacillus delbrueckii subsp. Bulgaricus, and is
+
−
                artificially synthesized based on the sequence retrieved from the GenBank.</p>
+
−
            <p>-GDH</p>
+
−
            <p>GDH (glucose dehydrogenase) catalyzes the conversion of beta-D-glucose to D-glucono-1,5-lactone and back,
+
−
                as it converts NADP+ to NADPH and back. It is used to catalyze the oxidation of beta-D-glucose into
+
−
                D-glucono-1,5-lactone and reduce NADP+ into NADPH during the process.</p>
+
−
            <p></p>
+
−
            <p>Source</p>
+
−
            <p>The GDH sequence is cloned from the genome of Bacillus subtilis through PCR amplification, using the
+
−
                primers designed and synthesized based on its sequence.</p>
+
−
            <p>-GFP</p>
+
−
            <p>Green fluorescent protein with his-tag.</p>
+
−
            <p></p>
+
−
            <p>Source</p>
+
−
            <p>The GFP DNA is artificially synthesized based on the sequence retrieved from the GenBank.</p>
+
−
            <p></p>
+
−
            <p>composite parts</p>
+
−
            <p>-ChBD</p>
+
−
            <p>--ChBD--7alpha-HSDH</p>
+
−
            <p>ChBD--7alpha-HSDH (T7 promoter--lac operator--RBS--His-tag--7alpha-HSDH--ChBD--T7 terminator)</p>
+
−
            <p></p>
+
−
            <p>This device codes for the 7alpha-HSDH--ChBD fusion protein. </p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Construct</h3></p>
+
−
            <p></p>
+
−
            <p>The vector of 7alpha-HSDH--ChBD for its expression is pET-28x. It is formed by modifying the restriction
+
−
                enzyme sites EcoR I and Xba I of vector pET-28a.</p>
+
−
            <p></p>
+
−
            <p>The 7alpha-HSDH sequence is cloned from the genome of E.coli DH5alpha through PCR amplification, using
+
−
                the primers designed and synthesized based on its sequence. The restriction site BamH I is added to the
+
−
                upstream primer, and Hind III is added to the downstream primer. </p>
+
−
            <p></p>
+
−
            <p>The ChBD sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid
+
−
                pUC57. The ChBD gene is then cloned from the plasmid by PCR amplification, with the restriction site
+
−
                Hind III added to the upstream primer, and Xhol I added to the downstream primer.</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>Firstly, the 7alpha-HSDH gene is inserted into the modified pET-28x at BamH I and Hind III, and ChBD at
+
−
                Hind III and Xhol I, after proliferation in T3 vector. Then the whole gene fragment, T7 promoter--lac
+
−
                operator--RBS--His-tag--7alpha-HSDH--ChBD--T7 terminator, is retrieved from this plasmid by PCR
+
−
                amplification, with prefix containing EcoR I, Not I and Xba I added on its upstream primer, and suffix
+
−
                containing Pst I, Not I and Spe I added on its downstream primer. The PCR product is then connected to
+
−
                pSB1C3 at EcoR I and Pst I.</p>
+
−
            <p></p>
+
−
            <p>[[file:psb1c3-chbd-7a.jpeg|300px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 1. pSB1C3--ChBD--7alpha-HSDH]</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Usage and Biology</h3></p>
+
−
            <p></p>
+
−
            <p>7alpha-HSDH(7alpha-hydroxysteroid dehydrogenase) catalyzes the oxidation of hydroxysteroids at C-7
+
−
                position and back, as it converts NAD+ to NADH and back. It catalyzes the oxidation of CDCA into
+
−
                intermediate product 7-oxo-LAC (7-ketolithocholic acid), where the hydroxyl at C-7 position becomes
+
−
                carbonyl.[1]</p>
+
−
            <p></p>
+
−
            <p>ChBD (chitin binding domain) is able to bind to chitin. When connected to 7alpha-HSDH, ChBD is able to
+
−
                immobilize the enzyme 7alpha-HSDH after expression, by binding to chitin powder inside the
+
−
                solution.[2]</p>
+
−
            <p></p>
+
−
            <p><h4>The function of chitin binding domain</h4></p>
+
−
            <p></p>
+
−
            <p>The function of ChBD is tested by connecting ChBD gene with GFP gene in pET28x. The GFP-ChBD fusion
+
−
                protein is expressed and mixed with chitin powder. Tube 3 contains GFP-ChBD, with a significant green
+
−
                color in the solution. After adding chitin powder inside the tube, the color in the solution disappears
+
−
                and shows on the chitin powder, as presented in tube 2. It proves that the ChBD successfully binds the
+
−
                GFP onto the chitin powder. In contrast, the color of the solution is not changed when adding chitin
+
−
                powder into the GFP solution without ChBD. As a result, the ChBD is well functioned. </p>
+
−
            <p></p>
+
−
            <p>[[file:chbd.jpeg|250px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig.2. ChBD function: tube1, GFP and chitin; tube2, GFP-ChBD and chitin; tube3, GFP-ChBD; tube4,
+
−
                water]</p>
+
−
            <p></p>
+
−
            <p><h4>Expression and Immobilization</h4></p>
+
−
            <p></p>
+
−
            <p>The constructed pET28x--7alpha-HSDH--ChBD plasmid is transformed into BL21(DE3) E.coli for expression.
+
−
                After that, when the OD 600 reached 0.6-0.8, 0.2mM IPTG is added in the liquid culture. The mixture is
+
−
                shaken at 20 ℃ overnight. The bacteria is collected by centrifugation at low temperature, 8000 rpm for
+
−
                10 minutes, and the supernatant is discarded. The bacteria is then resuspended using 0.15M pH8.8
+
−
                 Tris-HCL, and is broken by ultrasonication.</p>
+
−
            <p></p>
+
−
            <p>The resulted bacteria solution is diluted to a certain concentration and mixed thoroughly with chitin
+
−
                 powder. As a result, the ChBD protein binds to the chitin powder, and the enzyme is successfully
+
−
                immobilized.</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Reference</h3></p>
+
−
            <p>[1] Ming-Min Zheng, Ru-Feng Wang, Chun-Xiu Li, Jian-He Xu: Two-step enzymatic synthesis of
+
−
                ursodeoxycholic acid with a new 7β-hydroxysteroid dehydrogenase from Ruminococcus torques. Process
+
−
                Biochemistry, Elsevier, 2015.</p>
+
−
            <p></p>
+
−
            <p>[2] Takahisa Ikegami, Terumasa Okada, Masayuki Hashimoto, Shizuka Seino, Takeshi Watanabe, and Masahiro
+
−
                Shirakawa: Solution Structure of the Chitin-binding Domain of Bacillus circulans WL-12 Chitinase A1. The
+
−
                Journal Of Biological Chemistry, 2000.</p>
+
−
            <p>--ChBD--7beta-HSDH</p>
+
−
            <p>ChBD--7beta-HSDH (T7 promoter--lac operator--RBS--His-tag--7beta-HSDH--ChBD--T7 terminator)</p>
+
−
            <p></p>
+
−
            <p>This device codes for the 7beta-HSDH--ChBD fusion protein. </p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Construct</h3></p>
+
−
            <p></p>
+
−
            <p>The vector of 7beta-HSDH--ChBD for its expression is pET-28x. It is formed by modifying the restriction
+
−
                enzyme sites EcoR I and Xba I of vector pET-28a.</p>
+
−
            <p></p>
+
−
            <p>The 7beta-HSDH sequence is retrieved from the GenBank. It is artificially synthesized and inserted into
+
−
                plasmid pUC57. The 7beta-HSDH gene is then cloned from the plasmid by PCR amplification. The restriction
+
−
                site BamH I is added to the upstream primer, and Hind III is added to the downstream primer. </p>
+
−
            <p></p>
+
−
            <p>The ChBD sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid
+
−
                pUC57. The ChBD gene is then cloned from the plasmid by PCR amplification, with the restriction site
+
−
                Hind III added to the upstream primer, and Xhol I added to the downstream primer.</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>Firstly, the 7beta-HSDH gene is inserted into the modified pET-28x at BamH I and Hind III, and ChBD at
+
−
                Hind III and Xhol I, after proliferation in T3 vector. Then the whole gene fragment, T7 promoter--lac
+
−
                operator--RBS--His-tag--7beta-HSDH--ChBD--T7 terminator, is retrieved from this plasmid by PCR
+
−
                amplification, with prefix containing EcoR I, Not I and Xba I added on its upstream primer, and suffix
+
−
                containing Pst I, Not I and Spe I added on its downstream primer. The PCR product is then connected to
+
−
                pSB1C3 at EcoR I and Pst I.</p>
+
−
            <p></p>
+
−
            <p>[[file:psb1c3-chbd-7b.jpeg|300px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 1. pSB1C3--ChBD--7beta-HSDH]</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Usage and Biology</h3></p>
+
−
            <p></p>
+
−
            <p>7beta-HSDH(7beta-hydroxysteroid dehydrogenase) catalyzes the reduction of hydroxysteroids at C-7 position
+
−
                and back, as it converts NADPH to NDAP+ and back. It catalyzes the reduction of the intermediate product
+
−
                7-oxo-LAC (7-ketolithocholic acid) into UDCA, the final product, where the carbonyl at C-7 position
+
−
                becomes hydroxyl.[1]</p>
+
−
            <p></p>
+
−
            <p>ChBD (chitin binding domain) is able to bind to chitin. When connected to 7beta-HSDH, ChBD is able to
+
−
                immobilize the enzyme 7beta-HSDH after expression, by binding to chitin powder inside the
+
−
                solution.[2]</p>
+
−
            <p></p>
+
−
            <p><h4>The function of chitin binding domain</h4></p>
+
−
            <p></p>
+
−
            <p>The function of ChBD is tested by connecting ChBD gene with GFP gene in pET28x. The GFP-ChBD fusion
+
−
                protein is expressed and mixed with chitin powder. Tube 3 contains GFP-ChBD, with a significant green
+
−
                color in the solution. After adding chitin powder inside the tube, the color in the solution disappears
+
−
                and shows on the chitin powder, as presented in tube 2. It proves that the ChBD successfully binds the
+
−
                GFP onto the chitin powder. In contrast, the color of the solution is not changed when adding chitin
+
−
                powder into the GFP solution without ChBD. As a result, the ChBD is well functioned. </p>
+
−
            <p></p>
+
−
            <p>[[file:chbd.jpeg|250px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig.2. ChBD function: tube1, GFP and chitin; tube2, GFP-ChBD and chitin; tube3, GFP-ChBD; tube4,
+
−
                water]</p>
+
−
            <p></p>
+
−
            <p><h4>Expression and Immobilization</h4></p>
+
−
            <p></p>
+
−
            <p>The constructed pET28x--7beta-HSDH--ChBD plasmid is transformed into BL21(DE3) E.coli for expression.
+
−
                After that, when the OD 600 reached 0.6-0.8, 0.2mM IPTG is added in the liquid culture. The mixture is
+
−
                shaken at 20 ℃ overnight. The bacteria is collected by centrifugation at low temperature, 8000 rpm for
+
−
                10 minutes, and the supernatant is discarded. The bacteria is then resuspended using 0.15M pH8.8
+
−
                Tris-HCL, and is broken by ultrasonication.</p>
+
−
            <p></p>
+
−
            <p>The resulted bacteria solution is diluted to a certain concentration and mixed thoroughly with chitin
+
−
                powder. As a result, the ChBD protein binds to the chitin powder, and the enzyme is successfully
+
−
                immobilized.</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Reference</h3></p>
+
−
            <p>[1] Ming-Min Zheng, Ru-Feng Wang, Chun-Xiu Li, Jian-He Xu: Two-step enzymatic synthesis of
+
−
                ursodeoxycholic acid with a new 7β-hydroxysteroid dehydrogenase from Ruminococcus torques. Process
+
−
                Biochemistry, Elsevier, 2015.</p>
+
−
            <p></p>
+
−
            <p>[2] Takahisa Ikegami, Terumasa Okada, Masayuki Hashimoto, Shizuka Seino, Takeshi Watanabe, and Masahiro
+
−
                Shirakawa: Solution Structure of the Chitin-binding Domain of Bacillus circulans WL-12 Chitinase A1. The
+
−
                Journal Of Biological Chemistry, 2000.</p>
+
−
            <p>--ChBD-LDH</p>
+
−
            <p>ChBD-LDH (T7 promoter--lac operator--RBS--His-tag--LDH--ChBD--T7 terminator)</p>
+
−
            <p></p>
+
−
            <p>This device codes for the LDH-ChBD fusion protein. </p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Construct</h3></p>
+
−
            <p></p>
+
−
            <p>The vector of LDH-ChBD for its expression is pET-28x. It is formed by modifying the restriction enzyme
+
−
                sites EcoR I and Xba I of vector pET-28a.</p>
+
−
            <p></p>
+
−
            <p>The LDH sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid
+
−
                pUC57. The LDH gene is then cloned from the plasmid by PCR amplification. The restriction site BamH I is
+
−
                added to the upstream primer, and Hind III is added to the downstream primer.[1] </p>
+
−
            <p></p>
+
−
            <p>The ChBD sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid
+
−
                pUC57. The ChBD gene is then cloned from the plasmid by PCR amplification, with the restriction site
+
−
                Hind III added to the upstream primer, and Xhol I added to the downstream primer.[2]</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>Firstly, the LDH is inserted into the modified pET-28x at BamH I and Hind III, and ChBD at Hind III and
+
−
                Xhol I, after proliferation in T3 vector. Then the whole gene fragment, T7 promoter--lac
+
−
                operator--RBS--His-tag--LDH--ChBD--T7 terminator, is retrieved from this plasmid by PCR amplification,
+
−
                with prefix containing EcoR I, Not I and Xba I added on its upstream primer, and suffix containing Pst
+
−
                I, Not I and Spe I added on its downstream primer. The PCR product is then connected to pSB1C3 at EcoR I
+
−
                and Pst I.</p>
+
−
            <p></p>
+
−
            <p>[[file:psb1c3-chbd-ldh.jpeg|300px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 1. pSB1C3-ChBD-LDH]</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Usage and Biology</h3></p>
+
−
            <p></p>
+
−
            <p>LDH(lactate dehydrogenase) catalyzes the conversion of lactate to pyruvic acid and back, as it converts
+
−
                NAD+ to NADH and back. It is used to catalyze the reduction of pyruvate into lactate, with the presence
+
−
                of NADH, which is oxidized into NAD+ at the same time.[1]</p>
+
−
            <p></p>
+
−
            <p>ChBD (chitin binding domain) is able to bind to chitin. When connected to LDH, ChBD is able to immobilize
+
−
                the enzyme LDH after expression, by binding to chitin powder inside the solution.[2]</p>
+
−
            <p></p>
+
−
            <p><h4>The function of chitin binding domain</h4></p>
+
−
            <p></p>
+
−
            <p>The function of ChBD is tested by connecting ChBD gene with GFP gene in pET28x. The GFP-ChBD fusion
+
−
                protein is expressed and mixed with chitin powder. Tube 3 contains GFP-ChBD, with a significant green
+
−
                color in the solution. After adding chitin powder inside the tube, the color in the solution disappears
+
−
                and shows on the chitin powder, as presented in tube 2. It proves that the ChBD successfully binds the
+
−
                GFP onto the chitin powder. In contrast, the color of the solution is not changed when adding chitin
+
−
                powder into the GFP solution without ChBD. As a result, the ChBD is well functioned. </p>
+
−
            <p></p>
+
−
            <p>[[file:chbd.jpeg|250px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig.2. ChBD function: tube1, GFP and chitin; tube2, GFP-ChBD and chitin; tube3, GFP-ChBD; tube4,
+
−
                water]</p>
+
−
            <p></p>
+
−
            <p><h4>Expression and Immobilization</h4></p>
+
−
            <p></p>
+
−
            <p>The constructed pET28x-LDH-ChBD plasmid is transformed into BL21(DE3) E.coli for expression. After that,
+
−
                when the OD 600 reached 0.6-0.8, 0.2mM IPTG is added in the liquid culture. The mixture is shaken at 20
+
−
                ℃ overnight. The bacteria is collected by centrifugation at low temperature, 8000 rpm for 10 minutes,
+
−
                and the supernatant is discarded. The bacteria is then resuspended using 0.15M pH8.8 Tris-HCL, and is
+
−
                broken by ultrasonication.</p>
+
−
            <p></p>
+
−
            <p>The resulted bacteria solution is diluted to a certain concentration and mixed thoroughly with chitin
+
−
                powder. As a result, the ChBD protein binds to the chitin powder, and the enzyme is successfully
+
−
                immobilized.</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Reference</h3></p>
+
−
            <p>[1] Ming-Min Zheng, Ru-Feng Wang, Chun-Xiu Li, Jian-He Xu: Two-step enzymatic synthesis of
+
−
                ursodeoxycholic acid with a new 7β-hydroxysteroid dehydrogenase from Ruminococcus torques. Process
+
−
                Biochemistry, Elsevier, 2015.</p>
+
−
            <p></p>
+
−
            <p>[2] Takahisa Ikegami, Terumasa Okada, Masayuki Hashimoto, Shizuka Seino, Takeshi Watanabe, and Masahiro
+
−
                Shirakawa: Solution Structure of the Chitin-binding Domain of Bacillus circulans WL-12 Chitinase A1. The
+
−
                Journal Of Biological Chemistry, 2000.</p>
+
−
            <p>--ChBD-GDH</p>
+
−
            <p>ChBD-GDH (T7 promoter--lac operator--RBS--His-tag--GDH--ChBD--T7 terminator)</p>
+
−
            <p></p>
+
−
            <p>This device codes for the GDH-ChBD fusion protein. </p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Construct</h3></p>
+
−
            <p></p>
+
−
            <p>The vector of GDH-ChBD for its expression is pET-28x. It is formed by modifying the restriction enzyme
+
−
                sites EcoR I and Xba I of vector pET-28a.</p>
+
−
            <p></p>
+
−
            <p>The GDH sequence is cloned from the genome of Bacillus subtilis through PCR amplification, using the
+
−
                primers designed and synthesized based on its sequence. The restriction site BamH I is added to the
+
−
                upstream primer, and Hind III is added to the downstream primer. </p>
+
−
            <p></p>
+
−
            <p>The ChBD sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid
+
−
                pUC57. The ChBD gene is then cloned from the plasmid by PCR amplification, with the restriction site
+
−
                Hind III added to the upstream primer, and Xhol I added to the downstream primer.</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>Firstly, the GDH is inserted into the modified pET-28x at BamH I and Hind III, and ChBD at Hind III and
+
−
                Xhol I, after proliferation in T3 vector. Then the whole gene fragment, T7 promoter--lac
+
−
                operator--RBS--His-tag--GDH--ChBD--T7 terminator, is retrieved from this plasmid by PCR amplification,
+
−
                with prefix containing EcoR I, Not I and Xba I added on its upstream primer, and suffix containing Pst
+
−
                I, Not I and Spe I added on its downstream primer. The PCR product is then connected to pSB1C3 at EcoR I
+
−
                and Pst I.</p>
+
−
            <p></p>
+
−
            <p>[[file:psb1c3-chbd-gdh.jpeg|300px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 1. pSB1C3-ChBD-GDH]</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Usage and Biology</h3></p>
+
−
            <p></p>
+
−
            <p>GDH(glucose dehydrogenase) catalyzes the conversion of beta-D-glucose to D-glucono-1,5-lactone and back,
+
−
                as it converts NADP+ to NADPH and back. It is used to catalyze the oxidation of beta-D-glucose into
+
−
                D-glucono-1,5-lactone and reduce NADP+ into NADPH during the process.[1]</p>
+
−
            <p></p>
+
−
            <p>ChBD (chitin binding domain) is able to bind to chitin. When connected to GDH, ChBD is able to immobilize
+
−
                the enzyme GDH after expression, by binding to chitin powder inside the solution.[2]</p>
+
−
            <p></p>
+
−
            <p><h4>The function of chitin binding domain</h4></p>
+
−
            <p></p>
+
−
            <p>The function of ChBD is tested by connecting ChBD gene with GFP gene in pET28x. The GFP-ChBD fusion
+
−
                protein is expressed and mixed with chitin powder. Tube 3 contains GFP-ChBD, with a significant green
+
−
                color in the solution. After adding chitin powder inside the tube, the color in the solution disappears
+
−
                and shows on the chitin powder, as presented in tube 2. It proves that the ChBD successfully binds the
+
−
                GFP onto the chitin powder. In contrast, the color of the solution is not changed when adding chitin
+
−
                powder into the GFP solution without ChBD. As a result, the ChBD is well functioned. </p>
+
−
            <p></p>
+
−
            <p>[[file:chbd.jpeg|250px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig.2. ChBD function: tube1, GFP and chitin; tube2, GFP-ChBD and chitin; tube3, GFP-ChBD; tube4,
+
−
                water]</p>
+
−
            <p></p>
+
−
            <p><h4>Expression and Immobilization</h4></p>
+
−
            <p></p>
+
−
            <p>The constructed pET28x-GDH-ChBD plasmid is transformed into BL21(DE3) E.coli for expression. After that,
+
−
                when the OD 600 reached 0.6-0.8, 0.2mM IPTG is added in the liquid culture. The mixture is shaken at 20
+
−
                ℃ overnight. The bacteria is collected by centrifugation at low temperature, 8000 rpm for 10 minutes,
+
−
                and the supernatant is discarded. The bacteria is then resuspended using 0.15M pH8.8 Tris-HCL, and is
+
−
                broken by ultrasonication.</p>
+
−
            <p></p>
+
−
            <p>The resulted bacteria solution is diluted to a certain concentration and mixed thoroughly with chitin
+
−
                powder. As a result, the ChBD protein binds to the chitin powder, and the enzyme is successfully
+
−
                immobilized.</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Reference</h3></p>
+
−
            <p>[1] Ming-Min Zheng, Ru-Feng Wang, Chun-Xiu Li, Jian-He Xu: Two-step enzymatic synthesis of
+
−
                ursodeoxycholic acid with a new 7β-hydroxysteroid dehydrogenase from Ruminococcus torques. Process
+
−
                Biochemistry, Elsevier, 2015.</p>
+
−
            <p></p>
+
−
            <p>[2] Takahisa Ikegami, Terumasa Okada, Masayuki Hashimoto, Shizuka Seino, Takeshi Watanabe, and Masahiro
+
−
                Shirakawa: Solution Structure of the Chitin-binding Domain of Bacillus circulans WL-12 Chitinase A1. The
+
−
                Journal Of Biological Chemistry, 2000.</p>
+
−
            <p>--ChBD-GFP</p>
+
−
            <p>ChBD-GFP (T7 promoter--lac operator--RBS--His-tag--GFP--ChBD--T7 terminator)</p>
+
−
            <p></p>
+
−
            <p>This device codes for the GFP-ChBD fusion protein. </p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Construct</h3></p>
+
−
            <p></p>
+
−
            <p>The vector of GFP-ChBD for its expression is pET-28x. It is formed by modifying the restriction enzyme
+
−
                sites EcoR I and Xba I of vector pET-28a.</p>
+
−
            <p></p>
+
−
            <p>The GFP sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid
+
−
                pUC57. The GFP gene is then cloned from the plasmid by PCR amplification. The restriction site BamH I is
+
−
                added to the upstream primer, and Hind III is added to the downstream primer. </p>
+
−
            <p></p>
+
−
            <p>The ChBD sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid
+
−
                pUC57. The ChBD gene is then cloned from the plasmid by PCR amplification, with the restriction site
+
−
                Hind III added to the upstream primer, and Xhol I added to the downstream primer.</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>Firstly, the GFP is inserted into the modified pET-28x at BamH I and Hind III, and ChBD at Hind III and
+
−
                Xhol I, after proliferation in T3 vector. Then the whole gene fragment, T7 promoter--lac
+
−
                operator--RBS--His-tag--GFP--ChBD--T7 terminator, is retrieved from this plasmid by PCR amplification,
+
−
                with prefix containing EcoR I, Not I and Xba I added on its upstream primer, and suffix containing Pst
+
−
                I, Not I and Spe I added on its downstream primer. The PCR product is then connected to pSB1C3 at EcoR I
+
−
                and Pst I.</p>
+
−
            <p></p>
+
−
            <p>[[file:psb1c3-chbd-gfp.jpeg|300px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 1. pSB1C3-ChBD-GFP]</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Usage and Biology</h3></p>
+
−
            <p></p>
+
−
            <p>This part is used to test the function of ChBD (chitin binding domain), which is able to bind to
+
−
                chitin.[1]</p>
+
−
            <p></p>
+
−
            <p><h4>Expression</h4></p>
+
−
            <p></p>
+
−
            <p>The constructed pET28x-GFP-ChBD plasmid is transformed into BL21(DE3) E.coli for expression. After that,
+
−
                when the OD 600 reached 0.6-0.8, 0.2mM IPTG is added in the liquid culture. The mixture is shaken at 20
+
−
                ℃ overnight. The bacteria is collected by centrifugation at low temperature, 8000 rpm for 10 minutes,
+
−
                and the supernatant is discarded. The bacteria is then resuspended using 0.15M pH8.8 Tris-HCL, and is
+
−
                broken by ultrasonication.</p>
+
−
            <p></p>
+
−
            <p><h4>Proof of the ChBD function</h4></p>
+
−
            <p></p>
+
−
            <p>The resulted solution after expression is mixed with chitin powder. Also, the solution with GFP-ChBD but
+
−
                without chitin, solution with the presence of GFP and chitin, and water in comparison, are
+
−
                presented.</p>
+
−
            <p></p>
+
−
            <p>Tube 3 contains GFP-ChBD, with a significant green color in the solution. After adding chitin powder
+
−
                inside the tube, the color in the solution disappears and shows on the chitin powder, as presented in
+
−
                tube 2. It proves that the ChBD successfully binds the GFP onto the chitin powder. In contrast, the
+
−
                color of the solution is not changed when adding chitin powder into the GFP solution without ChBD. As a
+
−
                result, the ChBD is well functioned. </p>
+
−
            <p></p>
+
−
            <p>[[file:chbd.jpeg|250px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig.2. ChBD function: tube1, GFP and chitin; tube2, GFP-ChBD and chitin; tube3, GFP-ChBD; tube4,
+
−
                water]</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Reference</h3></p>
+
−
            <p>[1] Takahisa Ikegami, Terumasa Okada, Masayuki Hashimoto, Shizuka Seino, Takeshi Watanabe, and Masahiro
+
−
                Shirakawa: Solution Structure of the Chitin-binding Domain of Bacillus circulans WL-12 Chitinase A1. The
+
−
                Journal Of Biological Chemistry, 2000.</p>
+
−
            <p>-CBD</p>
+
−
            <p>--CBD--7alpha-HSDH</p>
+
−
            <p>CBD--7alpha-HSDH (T7 promoter--lac operator--RBS--His-tag--7alpha-HSDH--CBD--T7 terminator)</p>
+
−
            <p></p>
+
−
            <p>This device codes for the 7alpha-HSDH--CBD fusion protein. </p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Construct</h3></p>
+
−
            <p>The vector of 7alpha-HSDH--CBD for its expression is pET-28x. It is formed by modifying the restriction
+
−
                enzyme sites EcoR I and Xba I of vector pET-28a.</p>
+
−
            <p></p>
+
−
            <p>The 7alpha-HSDH sequence is cloned from the genome of E.coli DH5alpha through PCR amplification, using
+
−
                the primers designed and synthesized based on its sequence. The restriction site BamH I is added to the
+
−
                upstream primer, and Hind III is added to the downstream primer. </p>
+
−
            <p></p>
+
−
            <p>The CBD sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid
+
−
                pUC57. The CBD gene is then cloned from the plasmid by PCR amplification, with the restriction site Hind
+
−
                III added to the upstream primer, and Xhol I added to the downstream primer.</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>Firstly, the 7alpha-HSDH gene is inserted into the modified pET-28x at BamH I and Hind III, and CBD at
+
−
                Hind III and Xhol I, after proliferation in T3 vector. Then the whole gene fragment, T7 promoter--lac
+
−
                operator--RBS--His-tag--7alpha-HSDH--CBD--T7 terminator, is retrieved from this plasmid by PCR
+
−
                amplification, with prefix containing EcoR I, Not I and Xba I added on its upstream primer, and suffix
+
−
                containing Pst I, Not I and Spe I added on its downstream primer. The PCR product is then connected to
+
−
                pSB1C3 at EcoR I and Pst I.</p>
+
−
            <p></p>
+
−
            <p>[[file:Psb1c3-cbd-7a.jpeg|300px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 1. pSB1C3--CBD--7alpha-HSDH]</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Usage and Biology</h3></p>
+
−
            <p></p>
+
−
            <p>7alpha-HSDH(7alpha-hydroxysteroid dehydrogenase) catalyzes the oxidation of hydroxysteroids at C-7
+
−
                position and back, as it converts NAD+ to NADH and back. It catalyzes the oxidation of CDCA into
+
−
                intermediate product 7-oxo-LAC (7-ketolithocholic acid), where the hydroxyl at C-7 position becomes
+
−
                carbonyl.[1]</p>
+
−
            <p></p>
+
−
            <p>CBD (cellulose binding domain) is able to bind to cellulose. When connected to 7alpha-HSDH, CBD is able
+
−
                to immobilize the enzyme 7alpha-HSDH after expression, by binding to the gauze inside the solution on
+
−
                its cellulose.[2]</p>
+
−
            <p></p>
+
−
            <p><h4>The function of cellulose binding domain</h4></p>
+
−
            <p></p>
+
−
            <p>The function of CBD is tested by connecting CBD gene with GFP gene in pET28x. The GFP-CBD fusion protein
+
−
                is expressed and mixed with a gauze piece. The green fluorescent on the gauze is not significantly
+
−
                reduced after washing, proving that the CDB is well functioned. In comparison, no green fluorescent is
+
−
                left after washing the gauze mixed with GFP-ChBD (Chintin binding domain). </p>
+
−
            <p></p>
+
−
            <p>[[file:cbd-before.jpeg|150px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 2. GFP-CBD on gauze before washing]</p>
+
−
            <p></p>
+
−
            <p>[[file:cbd-after.jpeg|150px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 3. GFP-CBD on gauze after washing]</p>
+
−
            <p></p>
+
−
            <p>[[file:chbd-before.jpeg|150px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 4. GFP-ChBD on gauze before washing]</p>
+
−
            <p></p>
+
−
            <p>[[file:chbd-after.jpeg|150px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 5. GFP-ChBD on gauze after washing]</p>
+
−
            <p></p>
+
−
            <p><h4>Expression and Immobilization[2]</h4></p>
+
−
            <p></p>
+
−
            <p>The constructed pET28x--7alpha-HSDH--CBD plasmid is transformed into BL21(DE3) E.coli for expression.
+
−
                After that, when the OD 600 reached 0.6-0.8, 0.2mM IPTG is added in the liquid culture. The mixture is
+
−
                shaken at 20 ℃ overnight. The bacteria is collected by centrifugation at low temperature, 8000 rpm for
+
−
                10 minutes, and the supernatant is discarded. The bacteria is then resuspended using 0.15M pH8.8
+
−
                Tris-HCL, and is broken by ultrasonication.</p>
+
−
            <p></p>
+
−
            <p>The resulted bacteria solution is diluted to a certain concentration and mixed with gauze piece and the
+
−
                gauze piece is washed three times by ddH2O afterwards. As a result, the CBD protein binds to the
+
−
                cellulose on gauze, and the enzyme is successfully immobilized.</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Enzyme Activity</h3></p>
+
−
            <p></p>
+
−
            <p><h4>The oxidation of CDCA to 7-OXO-LCA using 7alpha-HSDH</h4></p>
+
−
            <p></p>
+
−
            <p>[[file:reaction1-1.jpeg|400px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 6. Reaction process]</p>
+
−
            <p></p>
+
−
            <p>7a-HSDH from Ecoli 5a is an NAD+ dependent enzyme. The 3 mL reaction consists of 150mM phosphate
+
−
                buffer(pH 8.0), 10mM CDCA, 0.2mM NAD+. The reaction started when the solution is combined with
+
−
                7a-HSDH-CBD-enzyme-binding gauze that in different concentration, includes 60ul, 120ul, and 180ul liquid
+
−
                supernatant of ultrasonication bacteria solution. The control group was testify under the same solution
+
−
                and condition but using pET28x-CBD liquid supernatant of ultrasonication bacteria solution to bind with
+
−
                gauze in the concentration of 180ul. Before adding the gauze into the solution, the gauze was washed by
+
−
                ddH2O for 3 times in order to purify the enzyme.</p>
+
−
            <p></p>
+
−
            <p>The CDCA was convert to 7OXO-LCA by loosing a pair of hydrogen(2H+and 2e-) from the 7-hydroxyl group and
+
−
                form 7-carbonyl group.The co-enzyme NAD+ is the acceptor of the a pair of hydrogen(2H+and 2e-) and e-,
+
−
                and was transformed into NADH. The enzyme activity was determined spectrophotometrically atv340 nm (ε =
+
−
                6.22 mM-1 cm-1) and room temperature. One unit of activity is defined as the amount of enzyme catalyzing
+
−
                the synthesize of 1 mmol of NADH per min under the assay conditions used.</p>
+
−
            <p></p>
+
−
            <p><h5>Result</h5></p>
+
−
            <p></p>
+
−
            <p>[[file:result1-1.jpeg|400px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 7. Result]</p>
+
−
            <p></p>
+
−
            <p><h4>Oxidation of CDCA to 7-oxo-LCA using E. coli 7alpha-HSDH and NAD+ regeneration</h4></p>
+
−
            <p></p>
+
−
            <p>[[file:reaction2-1.jpeg|400px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 8. Reaction process]</p>
+
−
            <p></p>
+
−
            <p>CDCA was converted in a 3mL solution containing 150 mM phosphate buffer(pH 8.0), 10 mM CDCA, 30 mM sodium
+
−
                pyruvate, 0.25mM NAD+, combined with 3U/ml LDH and1 1 U/ml E. coli DH5a 7a-HSDH-CBD at room
+
−
                temperature. </p>
+
−
            <p></p>
+
−
            <p>The bioconversion experiments were monitored via HPLC measurements. The sample was analyzed 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 chromatographic column C18.</p>
+
−
            <p></p>
+
−
            <p><h5>Result</h5></p>
+
−
            <p></p>
+
−
            <p>As shown in figure, the transformation was complete after 2.5h. </p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>[[file:hplc1-1.jpeg|500px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 9. Absorbance of NADH]</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>[[file:hplc1-2.jpeg|400px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 10. Peak area of 7oxo-LCA sample in different concentration]</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>[[file:hplc1-3.jpeg|500px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 11. Time course]</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <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></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>
+
−
            <p>
+
−
            <h3>Reference</h3></p>
+
−
            <p>[1] Ming-Min Zheng, Ru-Feng Wang, Chun-Xiu Li, Jian-He Xu: Two-step enzymatic synthesis of
+
−
                ursodeoxycholic acid with a new 7β-hydroxysteroid dehydrogenase from Ruminococcus torques. Process
+
−
                Biochemistry, Elsevier, 2015.</p>
+
−
            <p></p>
+
−
            <p>[2] Etai Shpigel, Arie Goldlust, Gilat Efroni, Amos Avraham, Adi Eshel, Mara Dekel, Oded Shoseyov:
+
−
                Immobilization of Recombinant Heparinase I Fused to Cellulose-Binding Domain, 1999.</p>
+
−
            <p>--CBD--7beta-HSDH</p>
+
−
            <p>CBD--7beta-HSDH (T7 promoter--lac operator--RBS--His-tag--7beta-HSDH--CBD--T7 terminator)</p>
+
−
            <p></p>
+
−
            <p>This device codes for the 7beta-HSDH--CBD fusion protein. </p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Construct</h3></p>
+
−
            <p></p>
+
−
            <p>The vector of 7beta-HSDH--CBD for its expression is pET-28x. It is formed by modifying the restriction
+
−
                enzyme sites EcoR I and Xba I of vector pET-28a.</p>
+
−
            <p></p>
+
−
            <p>The 7beta-HSDH sequence is retrieved from the GenBank. It is artificially synthesized and inserted into
+
−
                plasmid pUC57. The 7beta-HSDH gene is then cloned from the plasmid by PCR amplification. The restriction
+
−
                site BamH I is added to the upstream primer, and Hind III is added to the downstream primer. </p>
+
−
            <p></p>
+
−
            <p>The CBD sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid
+
−
                pUC57. The CBD gene is then cloned from the plasmid by PCR amplification, with the restriction site Hind
+
−
                III added to the upstream primer, and Xhol I added to the downstream primer.</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>Firstly, the 7beta-HSDH gene is inserted into the modified pET-28x at BamH I and Hind III, and CBD at
+
−
                Hind III and Xhol I, after proliferation in T3 vector. Then the whole gene fragment, T7 promoter--lac
+
−
                operator--RBS--His-tag--7beta-HSDH--CBD--T7 terminator, is retrieved from this plasmid by PCR
+
−
                amplification, with prefix containing EcoR I, Not I and Xba I added on its upstream primer, and suffix
+
−
                containing Pst I, Not I and Spe I added on its downstream primer. The PCR product is then connected to
+
−
                pSB1C3 at EcoR I and Pst I.</p>
+
−
            <p></p>
+
−
            <p>[[file:Psb1c3-cbd-7b.jpeg|300px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 1. pSB1C3--CBD--7beta-HSDH]</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Usage and Biology</h3></p>
+
−
            <p></p>
+
−
            <p>7beta-HSDH(7beta-hydroxysteroid dehydrogenase) catalyzes the reduction of hydroxysteroids at C-7 position
+
−
                and back, as it converts NADPH to NDAP+ and back. It catalyzes the reduction of the intermediate product
+
−
                7-oxo-LAC (7-ketolithocholic acid) into UDCA, the final product, where the carbonyl at C-7 position
+
−
                becomes hydroxyl.[1]</p>
+
−
            <p></p>
+
−
            <p>CBD (cellulose binding domain) is able to bind to cellulose. When connected to 7beta-HSDH, CBD is able to
+
−
                immobilize the enzyme 7beta-HSDH after expression, by binding to the gauze inside the solution on its
+
−
                cellulose.[2]</p>
+
−
            <p></p>
+
−
            <p><h4>The function of cellulose binding domain</h4></p>
+
−
            <p></p>
+
−
            <p>The function of CBD is tested by connecting CBD gene with GFP gene in pET28x. The GFP-CBD fusion protein
+
−
                is expressed and mixed with a gauze piece. The green fluorescent on the gauze is not significantly
+
−
                reduced after washing, proving that the CDB is well functioned. In comparison, no green fluorescent is
+
−
                left after washing the gauze mixed with GFP-ChBD (Chintin binding domain). </p>
+
−
            <p></p>
+
−
            <p>[[file:cbd-before.jpeg|150px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 2. GFP-CBD on gauze before washing]</p>
+
−
            <p></p>
+
−
            <p>[[file:cbd-after.jpeg|150px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 3. GFP-CBD on gauze after washing]</p>
+
−
            <p></p>
+
−
            <p>[[file:chbd-before.jpeg|150px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 4. GFP-ChBD on gauze before washing]</p>
+
−
            <p></p>
+
−
            <p>[[file:chbd-after.jpeg|150px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 5. GFP-ChBD on gauze after washing]</p>
+
−
            <p></p>
+
−
            <p><h4>Expression and Immobilization[2]</h4></p>
+
−
            <p></p>
+
−
            <p>The constructed pET28x--7beta-HSDH--CBD plasmid is transformed into BL21(DE3) E.coli for expression.
+
−
                After that, when the OD 600 reached 0.6-0.8, 0.2mM IPTG is added in the liquid culture. The mixture is
+
−
                shaken at 20 ℃ overnight. The bacteria is collected by centrifugation at low temperature, 8000 rpm for
+
−
                10 minutes, and the supernatant is discarded. The bacteria is then resuspended using 0.15M pH8.8
+
−
                Tris-HCL, and is broken by ultrasonication.</p>
+
−
            <p></p>
+
−
            <p>The resulted bacteria solution is diluted to a certain concentration and mixed with gauze piece, and the
+
−
                gauze piece is washed three times by ddH2O afterwards. As a result, the CBD protein binds to the
+
−
                cellulose on gauze, and the enzyme is successfully immobilized.</p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Enzyme Activity</h3></p>
+
−
            <p></p>
+
−
            <p><h4>The reduction of 7OXO-LCA to UDCA</h4></p>
+
−
            <p></p>
+
−
            <p>[[file:reaction1-2.jpeg|400px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 6. Reaction process]</p>
+
−
            <p></p>
+
−
            <p>7β-HSDH-CBD is an NADPH dependent enzyme from Ruminococcus Torques. The 3mL reaction contains 150mM
+
−
                phosphate buffer(pH 8.0), 10mM UDCA, 0.2mM NADPH. The reaction started when the solution is combined
+
−
                with 7β-HSDH-CBD-enzyme-binding gauze that in different concentration, includes 80ul, 120ul, 160ul
+
−
                liquid supernatant of ultrasonication bacteria solution. The control group was testify under the same
+
−
                solution and condition but using pET28x-CBD liquid supernatant of ultrasonication bacteria solution to
+
−
                bind with gauze in the concentration of 160ul. Before adding the gauze into the solution, the gauze was
+
−
                washed by ddH2O for 3 times in order to purify the enzyme.</p>
+
−
            <p></p>
+
−
            <p>The 7-OXO-LCA was convert to UDCA by Taking a a pair of hydrogen(2H+and 2e-)from the NADPH and form
+
−
                7-hydroxyl group (B position). The co-enzyme NADPH is the donor of the a pair of hydrogen(2H+and 2e-),
+
−
                and was transformed into NADP+. However, to make the reaction easy to testify, the reverse reaction that
+
−
                transforming UDCA to 7OXO-LCA was employed to prove the function of 7B in which The B position
+
−
                7-hydroxyl group loose a pair of hydrogen(2H+and 2e-) and from the 7-carbonyl group, and NADP+ is the
+
−
                acceptor of the a pair of hydrogen(2H+and 2e-), to form NADPH The the synthesized NADHP can be
+
−
                determined spectrophotometrically at340 nm (ε = 6.22 mM-1 cm-1) and room temperature. One unit of
+
−
                activity is defined as the amount of enzyme catalyzing the synthesize of 1 mmol of NADPH per min under
+
−
                the assay conditions used.</p>
+
−
            <p></p>
+
−
            <p><h5>Result</h5></p>
+
−
            <p></p>
+
−
            <p>[[file:result1-3.jpeg|400px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 7. Result]</p>
+
−
            <p></p>
+
−
            <p><h4>Reduction of 7-oxo-LCA to UDCA using 7β-HSDH and GDH(NADPH regeneration)</h4></p>
+
−
            <p></p>
+
−
            <p>[[file:reaction2-2.jpeg|400px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 8. Reaction process]</p>
+
−
            <p></p>
+
−
            <p>The 3mL reaction solution containing 150 mM phosphate buffer(pH 8.0), 10 mM UDCA, 30 mM glucose, 0.2mM
+
−
                NADP+, combined with 1U/ml 7β-HSDH and 5U/ml GDH at room temperature.</p>
+
−
            <p></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 chromatographic column C18
+
−
                .</p>
+
−
            <p></p>
+
−
            <p><h5>Result</h5></p>
+
−
            <p></p>
+
−
            <p>[[file:hplc2-1.jpeg|500px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 9. Absorbance of NADPH]</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>[[file:hplc2-2.jpeg|400px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 10. Peak area of 7oxo-LCA sample in different concentration]</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>[[file:hplc2-3.jpeg|500px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 11. Time course]</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <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></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>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Reference</h3></p>
+
−
            <p>[1] Ming-Min Zheng, Ru-Feng Wang, Chun-Xiu Li, Jian-He Xu: Two-step enzymatic synthesis of
+
−
                ursodeoxycholic acid with a new 7β-hydroxysteroid dehydrogenase from Ruminococcus torques. Process
+
−
                Biochemistry, Elsevier, 2015.</p>
+
−
            <p></p>
+
−
            <p>[2] Etai Shpigel, Arie Goldlust, Gilat Efroni, Amos Avraham, Adi Eshel, Mara Dekel, Oded Shoseyov:
+
−
                Immobilization of Recombinant Heparinase I Fused to Cellulose-Binding Domain, 1999.</p>
+
−
            <p>--CBD-LDH</p>
+
−
            <p>CBD-LDH (T7 promoter--lac operator--RBS--His-tag--LDH--CBD--T7 terminator)</p>
+
−
            <p></p>
+
−
            <p>This device codes for the LDH-CBD fusion protein. </p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Construct</h3></p>
+
−
            <p></p>
+
−
            <p>The vector of LDH-CBD for its expression is pET-28x. It is formed by modifying the restriction enzyme
+
−
                sites EcoR I and Xba I of vector pET-28a.</p>
+
−
            <p></p>
+
−
            <p>The LDH sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid
+
−
                pUC57. The LDH gene is then cloned from the plasmid by PCR amplification. The restriction site BamH I is
+
−
                added to the upstream primer, and Hind III is added to the downstream primer. </p>
+
−
            <p></p>
+
−
            <p>The CBD sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid
+
−
                pUC57. The CBD gene is then cloned from the plasmid by PCR amplification, with the restriction site Hind
+
−
                III added to the upstream primer, and Xhol I added to the downstream primer.</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>Firstly, the LDH gene is inserted into the modified pET-28x at BamH I and Hind III, and CBD at Hind III
+
−
                and Xhol I, after proliferation in T3 vector. Then the whole gene fragment, T7 promoter--lac
+
−
                operator--RBS--His-tag--LDH--CBD--T7 terminator, is retrieved from this plasmid by PCR amplification,
+
−
                with prefix containing EcoR I, Not I and Xba I added on its upstream primer, and suffix containing Pst
+
−
                I, Not I and Spe I added on its downstream primer. The PCR product is then connected to pSB1C3 at EcoR I
+
−
                and Pst I.</p>
+
−
            <p></p>
+
−
            <p>[[file:Psb1c3-cbd-ldh.jpeg|300px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 1. pSB1C3-CBD-LDH]</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Usage and Biology</h3></p>
+
−
            <p></p>
+
−
            <p>LDH(lactate dehydrogenase) catalyzes the conversion of lactate to pyruvic acid and back, as it converts
+
−
                NAD+ to NADH and back. It is used to catalyze the reduction of pyruvate into lactate, with the presence
+
−
                of NADH, which is oxidized into NAD+ at the same time.[1]</p>
+
−
            <p></p>
+
−
            <p>CBD (cellulose binding domain) is able to bind to cellulose. When connected to LDH, CBD is able to
+
−
                immobilize the enzyme LDH after expression, by binding to the gauze inside the solution on its
+
−
                cellulose.[2]</p>
+
−
            <p></p>
+
−
            <p><h4>The function of cellulose binding domain</h4></p>
+
−
            <p></p>
+
−
            <p>The function of CBD is tested by connecting CBD gene with GFP gene in pET28x. The GFP-CBD fusion protein
+
−
                is expressed and mixed with a gauze piece. The green fluorescent on the gauze is not significantly
+
−
                reduced after washing, proving that the CDB is well functioned. In comparison, no green fluorescent is
+
−
                left after washing the gauze mixed with GFP-ChBD (Chintin binding domain). </p>
+
−
            <p></p>
+
−
            <p>[[file:cbd-before.jpeg|150px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 2. GFP-CBD on gauze before washing]</p>
+
−
            <p></p>
+
−
            <p>[[file:cbd-after.jpeg|150px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 3. GFP-CBD on gauze after washing]</p>
+
−
            <p></p>
+
−
            <p>[[file:chbd-before.jpeg|150px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 4. GFP-ChBD on gauze before washing]</p>
+
−
            <p></p>
+
−
            <p>[[file:chbd-after.jpeg|150px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 5. GFP-ChBD on gauze after washing]</p>
+
−
            <p></p>
+
−
            <p><h4>Expression and Immobilization[2]</h4></p>
+
−
            <p></p>
+
−
            <p>The constructed pET28x-LDH plasmid is transformed into BL21(DE3) E.coli for expression. After that, when
+
−
                the OD 600 reached 0.6-0.8, 0.2mM IPTG is added in the liquid culture. The mixture is shaken at 20 ℃
+
−
                overnight. The bacteria is collected by centrifugation at low temperature, 8000 rpm for 10 minutes, and
+
−
                the supernatant is discarded. The bacteria is then resuspended using 0.15M pH8.8 Tris-HCL, and is broken
+
−
                by ultrasonication.</p>
+
−
            <p></p>
+
−
            <p>The resulted bacteria solution is diluted to a certain concentration and mixed with gauze piece and the
+
−
                gauze piece is washed three times by ddH2O afterwards. As a result, the CBD protein binds to the
+
−
                cellulose on gauze, and the enzyme is successfully immobilized.</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Enzyme Activity</h3></p>
+
−
            <p></p>
+
−
            <p><h4>The NAD+ regeneration</h4></p>
+
−
            <p></p>
+
−
            <p>LDH is an NADH dependent enzyme from Lactobacillus delbrueckii subspecies bulgaricus. The 3mL reaction
+
−
                consists of 150mM phosphate buffer(pH 8.0), 25mM Lactose, 0.2mM NAD+. The reaction started when the
+
−
                solution is combined with LDH-CBD-enzyme-binding gauze that in different concentration, includes 5ul,
+
−
                10ul, and 15ul liquid supernatant of ultrasonication bacteria solution. The control group was testify
+
−
                under the same solution and condition but using pET28x-CBD liquid supernatant of ultrasonication
+
−
                bacteria solution to bind with gauze in the concentration of 15ull. Before adding the gauze into the
+
−
                solution, the gauze was washed by ddH2O for 3 times in order to purify the enzyme.</p>
+
−
            <p></p>
+
−
            <p>The LDH works on pyruvate and take a pair of hydrogen(2H+and 2e-) from NADH to form lactate. The NAD+ was
+
−
                regenerate through this reaction. Since the NADH can be testified under 340nm of ultraviolet light, the
+
−
                reverse reaction of LDH is employed to testify its function. The enzyme activity was determined
+
−
                spectrophotometrically atv340 nm (ε = 6.22 mM-1 cm-1) and room temperature by measuring the synthesize
+
−
                of NADH. One unit of activity is defined as the amount of enzyme catalyzing the synthesize of 1 mmol of
+
−
                NADH per min under the assay conditions used.</p>
+
−
            <p></p>
+
−
            <p><h5>Result</h5></p>
+
−
            <p></p>
+
−
            <p>[[file:result1-2.jpeg|400px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 6. Result]</p>
+
−
            <p></p>
+
−
            <p><h4>Oxidation of CDCA to 7-oxo-LCA using E. coli 7alpha-HSDH and NAD+ regeneration</h4></p>
+
−
            <p></p>
+
−
            <p>[[file:reaction2-1.jpeg|400px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 7. Reaction process]</p>
+
−
            <p></p>
+
−
            <p>CDCA was converted in a 3mL solution containing 150 mM phosphate buffer(pH 8.0), 10 mM CDCA, 30 mM sodium
+
−
                pyruvate, 0.25mM NAD+, combined with 3U/ml LDH and1 1 U/ml E. coli DH5a 7a-HSDH-CBD at room
+
−
                temperature. </p>
+
−
            <p></p>
+
−
            <p>The bioconversion experiments were monitored via HPLC measurements. The sample was analyzed 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 chromatographic column C18.</p>
+
−
            <p></p>
+
−
            <p><h5>Result</h5></p>
+
−
            <p></p>
+
−
            <p>As shown in figure, the transformation was complete after 2.5h. </p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>[[file:hplc1-1.jpeg|500px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 9. Absorbance of NADH]</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>[[file:hplc1-2.jpeg|400px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 10. Peak area of 7oxo-LCA sample in different concentration]</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>[[file:hplc1-3.jpeg|500px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 11. Time course]</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <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></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>
+
−
            <p>
+
−
            <h3>Reference</h3></p>
+
−
            <p>[1] Ming-Min Zheng, Ru-Feng Wang, Chun-Xiu Li, Jian-He Xu: Two-step enzymatic synthesis of
+
−
                ursodeoxycholic acid with a new 7β-hydroxysteroid dehydrogenase from Ruminococcus torques. Process
+
−
                Biochemistry, Elsevier, 2015.</p>
+
−
            <p></p>
+
−
            <p>[2] Etai Shpigel, Arie Goldlust, Gilat Efroni, Amos Avraham, Adi Eshel, Mara Dekel, Oded Shoseyov:
+
−
                Immobilization of Recombinant Heparinase I Fused to Cellulose-Binding Domain, 1999.</p>
+
−
            <p></p>
+
−
            <p>--CBD-GDH</p>
+
−
            <p>CBD-GDH (T7 promoter--lac operator--RBS--His-tag--GDH--CBD--T7 terminator)</p>
+
−
            <p></p>
+
−
            <p>This device codes for the GDH-CBD fusion protein. </p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Construct</h3></p>
+
−
            <p></p>
+
−
            <p>The vector of GDH-CBD for its expression is pET-28x. It is formed by modifying the restriction enzyme
+
−
                sites EcoR I and Xba I of vector pET-28a.</p>
+
−
            <p></p>
+
−
            <p>The GDH sequence is cloned from the genome of Bacillus subtilis through PCR amplification, using the
+
−
                primers designed and synthesized based on its sequence. The restriction site BamH I is added to the
+
−
                upstream primer, and Hind III is added to the downstream primer. </p>
+
−
            <p></p>
+
−
            <p>The CBD sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid
+
−
                pUC57. The CBD gene is then cloned from the plasmid by PCR amplification, with the restriction site Hind
+
−
                III added to the upstream primer, and Xhol I added to the downstream primer.</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>Firstly, the GDH gene is inserted into the modified pET-28x at BamH I and Hind III, and CBD at Hind III
+
−
                and Xhol I, after proliferation in T3 vector. Then the whole gene fragment, T7 promoter--lac
+
−
                operator--RBS--His-tag--GDH--CBD--T7 terminator, is retrieved from this plasmid by PCR amplification,
+
−
                with prefix containing EcoR I, Not I and Xba I added on its upstream primer, and suffix containing Pst
+
−
                I, Not I and Spe I added on its downstream primer. The PCR product is then connected to pSB1C3 at EcoR I
+
−
                and Pst I.</p>
+
−
            <p></p>
+
−
            <p>[[file:Psb1c3-cbd-gdh.jpeg|300px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 1. pSB1C3-CBD-GDH]</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Usage and Biology</h3></p>
+
−
            <p></p>
+
−
            <p>GDH(glucose dehydrogenase) catalyzes the conversion of beta-D-glucose to D-glucono-1,5-lactone and back,
+
−
                as it converts NADP+ to NADPH and back. It is used to catalyze the oxidation of beta-D-glucose into
+
−
                D-glucono-1,5-lactone and reduce NADP+ into NADPH during the process.[1]</p>
+
−
            <p></p>
+
−
            <p>CBD (cellulose binding domain) is able to bind to cellulose. When connected to GDH, CBD is able to
+
−
                immobilize the enzyme GDH after expression, by binding to the gauze inside the solution on its
+
−
                cellulose.[2]</p>
+
−
            <p></p>
+
−
            <p><h4>The function of cellulose binding domain</h4></p>
+
−
            <p></p>
+
−
            <p>The function of CBD is tested by connecting CBD gene with GFP gene in pET28x. The GFP-CBD fusion protein
+
−
                is expressed and mixed with a gauze piece. The green fluorescent on the gauze is not significantly
+
−
                reduced after washing, proving that the CDB is well functioned. In comparison, no green fluorescent is
+
−
                left after washing the gauze mixed with GFP-ChBD (Chintin binding domain). </p>
+
−
            <p></p>
+
−
            <p>[[file:cbd-before.jpeg|150px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 2. GFP-CBD on gauze before washing]</p>
+
−
            <p></p>
+
−
            <p>[[file:cbd-after.jpeg|150px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 3. GFP-CBD on gauze after washing]</p>
+
−
            <p></p>
+
−
            <p>[[file:chbd-before.jpeg|150px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 4. GFP-ChBD on gauze before washing]</p>
+
−
            <p></p>
+
−
            <p>[[file:chbd-after.jpeg|150px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 5. GFP-ChBD on gauze after washing]</p>
+
−
            <p></p>
+
−
            <p><h4>Expression and Immobilization[2]</h4></p>
+
−
            <p></p>
+
−
            <p>The constructed pET28x-GDH plasmid is transformed into BL21(DE3) E.coli for expression. After that, when
+
−
                the OD 600 reached 0.6-0.8, 0.2mM IPTG is added in the liquid culture. The mixture is shaken at 20 ℃
+
−
                overnight. The bacteria is collected by centrifugation at low temperature, 8000 rpm for 10 minutes, and
+
−
                the supernatant is discarded. The bacteria is then resuspended using 0.15M pH8.8 Tris-HCL, and is broken
+
−
                by ultrasonication.</p>
+
−
            <p></p>
+
−
            <p>The resulted bacteria solution is diluted to a certain concentration and mixed with gauze piece and the
+
−
                gauze piece is washed three times by ddH2O afterwards. As a result, the CBD protein binds to the
+
−
                cellulose on gauze, and the enzyme is successfully immobilized.</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Enzyme Activity</h3></p>
+
−
            <p></p>
+
−
            <p><h4>Regeneration of NADPH</h4></p>
+
−
            <p></p>
+
−
            <p>GDH is an NADPH dependent enzyme from bacillus subtilis. The 3mL reaction consists of 150mM phosphate
+
−
                buffer(pH 6.5), 30mM glucose, 0.2mM NADP+. The reaction started when the solution is combined with
+
−
                GDH-CBD-enzyme-binding gauze that in different concentration, includes 80ul, 120ul, and 160ul liquid
+
−
                supernatant of ultrasonication bacteria solution. The control group was testify under the same solution
+
−
                and condition but using pET28x-CBD liquid supernatant of ultrasonication bacteria solution to bind with
+
−
                gauze in the concentration of 160ul. Before adding the gauze into the solution, the gauze was washed by
+
−
                ddH2O for 3 times in order to purify the enzyme.</p>
+
−
            <p></p>
+
−
            <p>The GDH works on glucose and take a pair of hydrogen(2H+and 2e-) from glucose and add to NADP+. The NADPH
+
−
                was regenerate through this reaction. Since the NADPH can be testified under 340nm of ultraviolet light,
+
−
                the enzyme activity was determined spectrophotometrically atv340 nm (ε = 6.22 mM-1 cm-1) and room
+
−
                temperature by measuring the synthesize of N. One unit of activity is defined as the amount of enzyme
+
−
                catalyzing the synthesize of 1 mmol of NADH per min under the assay conditions used.</p>
+
−
            <p></p>
+
−
            <p><h5>Result</h5></p>
+
−
            <p></p>
+
−
            <p>[[file:result1-4.jpeg|400px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 6. Result]</p>
+
−
            <p></p>
+
−
            <p><h4>Reduction of 7-oxo-LCA to UDCA using 7β-HSDH and GDH(NADPH regeneration)</h4></p>
+
−
            <p></p>
+
−
            <p>[[file:reaction2-2.jpeg|400px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 7. Reaction process]</p>
+
−
            <p></p>
+
−
            <p>The 3mL reaction solution containing 150 mM phosphate buffer(pH 8.0), 10 mM UDCA, 30 mM glucose, 0.2mM
+
−
                 NADP+, combined with 1U/ml 7β-HSDH and 5U/ml GDH at room temperature.</p>
+
−
            <p></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 chromatographic column C18
+
−
                .</p>
+
−
            <p></p>
+
−
            <p><h5>Result</h5></p>
+
−
            <p>[[file:hplc2-1.jpeg|500px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 9. Absorbance of NADPH]</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>[[file:hplc2-2.jpeg|400px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 10. Peak area of 7oxo-LCA sample in different concentration]</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>[[file:hplc2-3.jpeg|500px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 11. Time course]</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <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></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>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Reference</h3></p>
+
−
            <p>[1] Ming-Min Zheng, Ru-Feng Wang, Chun-Xiu Li, Jian-He Xu: Two-step enzymatic synthesis of
+
−
                ursodeoxycholic acid with a new 7β-hydroxysteroid dehydrogenase from Ruminococcus torques. Process
+
−
                Biochemistry, Elsevier, 2015.</p>
+
−
            <p></p>
+
−
            <p>[2] Etai Shpigel, Arie Goldlust, Gilat Efroni, Amos Avraham, Adi Eshel, Mara Dekel, Oded Shoseyov:
+
−
                Immobilization of Recombinant Heparinase I Fused to Cellulose-Binding Domain, 1999.</p>
+
−
            <p></p>
+
−
            <p>--CBD-GFP</p>
+
−
            <p>CBD-GFP (T7 promoter--lac operator--RBS--His-tag--GFP--CBD--T7 terminator)</p>
+
−
            <p></p>
+
−
            <p>This device codes for the GFP-CBD fusion protein. </p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Construct</h3></p>
+
−
            <p></p>
+
−
            <p>The vector of GFP-CBD for its expression is pET-28x. It is formed by modifying the restriction enzyme
+
−
                sites EcoR I and Xba I of vector pET-28a.</p>
+
−
            <p></p>
+
−
            <p>The GFP sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid
+
−
                pUC57. The GFP gene is then cloned from the plasmid by PCR amplification. The restriction site BamH I is
+
−
                added to the upstream primer, and Hind III is added to the downstream primer. </p>
+
−
            <p></p>
+
−
            <p>The CBD sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid
+
−
                pUC57. The CBD gene is then cloned from the plasmid by PCR amplification, with the restriction site Hind
+
−
                III added to the upstream primer, and Xhol I added to the downstream primer.</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>Firstly, the GFP gene is inserted into the modified pET-28x at BamH I and Hind III, and CBD at Hind III
+
−
                and Xhol I, after proliferation in T3 vector. Then the whole gene fragment, T7 promoter--lac
+
−
                operator--RBS--His-tag--GFP--CBD--T7 terminator, is retrieved from this plasmid by PCR amplification,
+
−
                with prefix containing EcoR I, Not I and Xba I added on its upstream primer, and suffix containing Pst
+
−
                I, Not I and Spe I added on its downstream primer. The PCR product is then connected to pSB1C3 at EcoR I
+
−
                and Pst I.</p>
+
−
            <p></p>
+
−
            <p>[[file:Psb1c3-cbd-gfp.jpeg|300px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 1. pSB1C3-CBD-GFP]</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Usage and Biology</h3></p>
+
−
            <p></p>
+
−
            <p>This part is used to test the function of CBD (cellulose binding domain), which is able to bind to
+
−
                cellulose.[1]</p>
+
−
            <p></p>
+
−
            <p><h4>Expression</h4></p>
+
−
            <p></p>
+
−
            <p>The constructed pET28x-GFP-CBD plasmid is transformed into BL21(DE3) E.coli for expression. After that,
+
−
                when the OD 600 reached 0.6-0.8, 0.2mM IPTG is added in the liquid culture. The mixture is shaken at 20
+
−
                ℃ overnight. The bacteria is collected by centrifugation at low temperature, 8000 rpm for 10 minutes,
+
−
                and the supernatant is discarded. The bacteria is then resuspended using 0.15M pH8.8 Tris-HCL, and is
+
−
                broken by ultrasonication.</p>
+
−
            <p></p>
+
−
            <p><h4>Proof of the CBD function</h4></p>
+
−
            <p></p>
+
−
            <p>The resulted solution after expression is mixed with a gauze piece, and the green color on gauze is
+
−
                recorded. Then the gauze is washed three times using ddH2O, but the green fluorescent on it is not
+
−
                significantly reduced, proving that the CDB is well functioned. In comparison, no green fluorescent is
+
−
                left after washing the gauze mixed with GFP-ChBD (Chintin binding domain). </p>
+
−
            <p></p>
+
−
            <p>[[file:cbd-before.jpeg|150px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 2. GFP-CBD on gauze before washing]</p>
+
−
            <p></p>
+
−
            <p>[[file:cbd-after.jpeg|150px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 3. GFP-CBD on gauze after washing]</p>
+
−
            <p></p>
+
−
            <p>[[file:chbd-before.jpeg|150px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 4. GFP-ChBD on gauze before washing]</p>
+
−
            <p></p>
+
−
            <p>[[file:chbd-after.jpeg|150px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 5. GFP-ChBD on gauze after washing]</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>
+
−
            <h3>Reference</h3></p>
+
−
            <p>[1] Etai Shpigel, Arie Goldlust, Gilat Efroni, Amos Avraham, Adi Eshel, Mara Dekel, Oded Shoseyov:
+
−
                Immobilization of Recombinant Heparinase I Fused to Cellulose-Binding Domain, 1999.</p>
+
−
            <p></p>
+
−
            <p></p>
+
−
            <p>contribution</p>
+
−
            <p>We improved the description of ChBD and CBD on previous part pages.</p>
+
−
            <p>-ChBD (Part:BBa_T2028)</p>
+
−
            <p></p>
+
−
            <p><h4>Proof of the ChBD function</h4></p>
+
−
            <p></p>
+
−
            <p>The resulted solution after expression is mixed with chitin powder. Also, the solution with GFP-ChBD but
+
−
                without chitin, solution with the presence of GFP and chitin, and water in comparison, are
+
−
                presented.</p>
+
−
            <p></p>
+
−
            <p>Tube 3 contains GFP-ChBD, with a significant green color in the solution. After adding chitin powder
+
−
                inside the tube, the color in the solution disappears and shows on the chitin powder, as presented in
+
−
                tube 2. It proves that the ChBD successfully binds the GFP onto the chitin powder. In contrast, the
+
−
                color of the solution is not changed when adding chitin powder into the GFP solution without ChBD. As a
+
−
                result, the ChBD is well functioned. </p>
+
−
            <p></p>
+
−
            <p>[[file:chbd.jpeg|250px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig.2. ChBD function: tube1, GFP and chitin; tube2, GFP-ChBD and chitin; tube3, GFP-ChBD; tube4,
+
−
                water]</p>
+
−
            <p></p>
+
−
            <p>-CBD (Part:BBa_K1979001)</p>
+
−
            <p></p>
+
−
            <p><h4>Proof of the CBD function</h4></p>
+
−
            <p></p>
+
−
            <p>The resulted solution after expression is mixed with a gauze piece, and the green color on gauze is
+
−
                recorded. Then the gauze is washed three times using ddH2O, but the green fluorescent on it is not
+
−
                significantly reduced, proving that the CDB is well functioned. In comparison, no green fluorescent is
+
−
                left after washing the gauze mixed with GFP-ChBD (Chintin binding domain). </p>
+
−
            <p></p>
+
−
            <p>[[file:cbd-before.jpeg|150px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 2. GFP-CBD on gauze before washing]</p>
+
−
            <p></p>
+
−
            <p>[[file:cbd-after.jpeg|150px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 3. GFP-CBD on gauze after washing]</p>
+
−
            <p></p>
+
−
            <p>[[file:chbd-before.jpeg|150px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 4. GFP-ChBD on gauze before washing]</p>
+
−
            <p></p>
+
−
            <p>[[file:chbd-after.jpeg|150px]]</p>
+
−
            <p></p>
+
−
            <p>[Fig. 5. GFP-ChBD on gauze after washing]</p>
+
−
            <p></p>
+
  
 +
                <!-- ROW -->
 +
 +
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                <tr>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td><a class="noul_link part_link"
 +
                          href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2239001">BBa_K2239001</a></td>
 +
                    <td>Generator</td>
 +
                    <td>ChBD-7alphaHSDH</td>
 +
                    <td width="100px">Ruohong Wang</td>
 +
                    <td align="right">1089</td>
 +
                    <td class="blank_col"></td>
 +
 +
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 +
                          action="/cgi/partsdb/pgroup.cgi?pgroup=iGEM2017&amp;group=SDSZ-China&amp;from=message&amp;isappinstalled=0"
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                    <input type="hidden" name="Editing" value="Editing"><input type="hidden" name="part"
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 +
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 +
                <tr>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td><a class="noul_link part_link"
 +
                          href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2239002">BBa_K2239002</a></td>
 +
                    <td>Generator</td>
 +
                    <td>ChBD-7betaHSDH</td>
 +
                    <td width="100px">Ruohong Wang</td>
 +
                    <td align="right">1179</td>
 +
                    <td class="blank_col"></td>
 +
 +
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 +
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                    <input type="hidden" name="Editing" value="Editing"><input type="hidden" name="part"
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                                                                              value="43613"><input type="hidden"
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 +
                                                                                                    value="iGEM2017"><input
 +
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 +
                <tr>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td><a class="noul_link part_link"
 +
                          href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2239003">BBa_K2239003</a></td>
 +
                    <td>Generator</td>
 +
                    <td>ChBD-LDH</td>
 +
                    <td width="100px">Ruohong Wang</td>
 +
                    <td align="right">1323</td>
 +
                    <td class="blank_col"></td>
 +
 +
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 +
                          action="/cgi/partsdb/pgroup.cgi?pgroup=iGEM2017&amp;group=SDSZ-China&amp;from=message&amp;isappinstalled=0"
 +
                          enctype="multipart/form-data"></form>
 +
                    <input type="hidden" name="Editing" value="Editing"><input type="hidden" name="part"
 +
                                                                              value="43616"><input type="hidden"
 +
                                                                                                    name="pgroup"
 +
                                                                                                    value="iGEM2017"><input
 +
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 +
                <tr>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td><a class="noul_link part_link"
 +
                          href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2239004">BBa_K2239004</a></td>
 +
                    <td>Generator</td>
 +
                    <td>ChBD-GDH</td>
 +
                    <td width="100px">Ruohong Wang</td>
 +
                    <td align="right">1107</td>
 +
                    <td class="blank_col"></td>
 +
 +
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 +
                          action="/cgi/partsdb/pgroup.cgi?pgroup=iGEM2017&amp;group=SDSZ-China&amp;from=message&amp;isappinstalled=0"
 +
                          enctype="multipart/form-data"></form>
 +
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 +
                                                                                                    value="iGEM2017"><input
 +
                        type="hidden" name="group" value="SDSZ-China"></tr>
 +
                <tr>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td><a class="noul_link part_link"
 +
                          href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2239005">BBa_K2239005</a></td>
 +
                    <td>Generator</td>
 +
                    <td>ChBD-GFP</td>
 +
                    <td width="100px">Ruohong Wang</td>
 +
                    <td align="right">1047</td>
 +
                    <td class="blank_col"></td>
 +
 +
                    <form method="post"
 +
                          action="/cgi/partsdb/pgroup.cgi?pgroup=iGEM2017&amp;group=SDSZ-China&amp;from=message&amp;isappinstalled=0"
 +
                          enctype="multipart/form-data"></form>
 +
                    <input type="hidden" name="Editing" value="Editing"><input type="hidden" name="part"
 +
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 +
                                                                                                    name="pgroup"
 +
                                                                                                    value="iGEM2017"><input
 +
                        type="hidden" name="group" value="SDSZ-China"></tr>
 +
                <tr>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td><a class="noul_link part_link"
 +
                          href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2239006">BBa_K2239006</a></td>
 +
                    <td>Generator</td>
 +
                    <td>CBD-7alphaHSDH</td>
 +
                    <td width="100px">Ruohong Wang</td>
 +
                    <td align="right">1080</td>
 +
                    <td class="blank_col"></td>
 +
 +
                    <form method="post"
 +
                          action="/cgi/partsdb/pgroup.cgi?pgroup=iGEM2017&amp;group=SDSZ-China&amp;from=message&amp;isappinstalled=0"
 +
                          enctype="multipart/form-data"></form>
 +
                    <input type="hidden" name="Editing" value="Editing"><input type="hidden" name="part"
 +
                                                                              value="43641"><input type="hidden"
 +
                                                                                                    name="pgroup"
 +
                                                                                                    value="iGEM2017"><input
 +
                        type="hidden" name="group" value="SDSZ-China"></tr>
 +
                <tr>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td><a class="noul_link part_link"
 +
                          href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2239007">BBa_K2239007</a></td>
 +
                    <td>Generator</td>
 +
                    <td>CBD-7betaHSDH</td>
 +
                    <td width="100px">Ruohong Wang</td>
 +
                    <td align="right">1170</td>
 +
                    <td class="blank_col"></td>
 +
 +
                    <form method="post"
 +
                          action="/cgi/partsdb/pgroup.cgi?pgroup=iGEM2017&amp;group=SDSZ-China&amp;from=message&amp;isappinstalled=0"
 +
                          enctype="multipart/form-data"></form>
 +
                    <input type="hidden" name="Editing" value="Editing"><input type="hidden" name="part"
 +
                                                                              value="43642"><input type="hidden"
 +
                                                                                                    name="pgroup"
 +
                                                                                                    value="iGEM2017"><input
 +
                        type="hidden" name="group" value="SDSZ-China"></tr>
 +
                <tr>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td><a class="noul_link part_link"
 +
                          href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2239008">BBa_K2239008</a></td>
 +
                    <td>Generator</td>
 +
                    <td>CBD-LDH</td>
 +
                    <td width="100px">Ruohong Wang</td>
 +
                    <td align="right">1314</td>
 +
                    <td class="blank_col"></td>
 +
 +
                    <form method="post"
 +
                          action="/cgi/partsdb/pgroup.cgi?pgroup=iGEM2017&amp;group=SDSZ-China&amp;from=message&amp;isappinstalled=0"
 +
                          enctype="multipart/form-data"></form>
 +
                    <input type="hidden" name="Editing" value="Editing"><input type="hidden" name="part"
 +
                                                                              value="43643"><input type="hidden"
 +
                                                                                                    name="pgroup"
 +
                                                                                                    value="iGEM2017"><input
 +
                        type="hidden" name="group" value="SDSZ-China"></tr>
 +
                <tr>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td><a class="noul_link part_link"
 +
                          href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2239009">BBa_K2239009</a></td>
 +
                    <td>Generator</td>
 +
                    <td>CBD-GDH</td>
 +
                    <td width="100px">Ruohong Wang</td>
 +
                    <td align="right">1098</td>
 +
                    <td class="blank_col"></td>
 +
 +
                    <form method="post"
 +
                          action="/cgi/partsdb/pgroup.cgi?pgroup=iGEM2017&amp;group=SDSZ-China&amp;from=message&amp;isappinstalled=0"
 +
                          enctype="multipart/form-data"></form>
 +
                    <input type="hidden" name="Editing" value="Editing"><input type="hidden" name="part"
 +
                                                                              value="43644"><input type="hidden"
 +
                                                                                                    name="pgroup"
 +
                                                                                                    value="iGEM2017"><input
 +
                        type="hidden" name="group" value="SDSZ-China"></tr>
 +
                <tr>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td><a class="noul_link part_link"
 +
                          href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2239010">BBa_K2239010</a></td>
 +
                    <td>Generator</td>
 +
                    <td>CBD-GFP</td>
 +
                    <td width="100px">Ruohong Wang</td>
 +
                    <td align="right">1038</td>
 +
                    <td class="blank_col"></td>
 +
 +
                    <form method="post"
 +
                          action="/cgi/partsdb/pgroup.cgi?pgroup=iGEM2017&amp;group=SDSZ-China&amp;from=message&amp;isappinstalled=0"
 +
                          enctype="multipart/form-data"></form>
 +
                    <input type="hidden" name="Editing" value="Editing"><input type="hidden" name="part"
 +
                                                                              value="43571"><input type="hidden"
 +
                                                                                                    name="pgroup"
 +
                                                                                                    value="iGEM2017"><input
 +
                        type="hidden" name="group" value="SDSZ-China"></tr>
 +
                <tr>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td><a class="noul_link part_link"
 +
                          href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2239011">BBa_K2239011</a></td>
 +
                    <td>Coding</td>
 +
                    <td>7alpha-HSDH</td>
 +
                    <td width="100px">Ruohong Wang</td>
 +
                    <td align="right">780</td>
 +
                    <td class="blank_col"></td>
 +
 +
                    <form method="post"
 +
                          action="/cgi/partsdb/pgroup.cgi?pgroup=iGEM2017&amp;group=SDSZ-China&amp;from=message&amp;isappinstalled=0"
 +
                          enctype="multipart/form-data"></form>
 +
                    <input type="hidden" name="Editing" value="Editing"><input type="hidden" name="part"
 +
                                                                              value="43574"><input type="hidden"
 +
                                                                                                    name="pgroup"
 +
                                                                                                    value="iGEM2017"><input
 +
                        type="hidden" name="group" value="SDSZ-China"></tr>
 +
                <tr>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td><a class="noul_link part_link"
 +
                          href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2239012">BBa_K2239012</a></td>
 +
                    <td>Coding</td>
 +
                    <td>7beta-HSDH</td>
 +
                    <td width="100px">Ruohong Wang</td>
 +
                    <td align="right">870</td>
 +
                    <td class="blank_col"></td>
 +
 +
                    <form method="post"
 +
                          action="/cgi/partsdb/pgroup.cgi?pgroup=iGEM2017&amp;group=SDSZ-China&amp;from=message&amp;isappinstalled=0"
 +
                          enctype="multipart/form-data"></form>
 +
                    <input type="hidden" name="Editing" value="Editing"><input type="hidden" name="part"
 +
                                                                              value="43701"><input type="hidden"
 +
                                                                                                    name="pgroup"
 +
                                                                                                    value="iGEM2017"><input
 +
                        type="hidden" name="group" value="SDSZ-China"></tr>
 +
                <tr>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td><a class="noul_link part_link"
 +
                          href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2239013">BBa_K2239013</a></td>
 +
                    <td>Coding</td>
 +
                    <td>GFP</td>
 +
                    <td width="100px">Ruohong Wang</td>
 +
                    <td align="right">738</td>
 +
                    <td class="blank_col"></td>
 +
 +
                    <form method="post"
 +
                          action="/cgi/partsdb/pgroup.cgi?pgroup=iGEM2017&amp;group=SDSZ-China&amp;from=message&amp;isappinstalled=0"
 +
                          enctype="multipart/form-data"></form>
 +
                    <input type="hidden" name="Editing" value="Editing"><input type="hidden" name="part"
 +
                                                                              value="43579"><input type="hidden"
 +
                                                                                                    name="pgroup"
 +
                                                                                                    value="iGEM2017"><input
 +
                        type="hidden" name="group" value="SDSZ-China"></tr>
 +
                <tr>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td><a class="noul_link part_link"
 +
                          href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2239014">BBa_K2239014</a></td>
 +
                    <td>Coding</td>
 +
                    <td>GDH (glucose dehydrogenas)</td>
 +
                    <td width="100px">Ruohong Wang</td>
 +
                    <td align="right">798</td>
 +
                    <td class="blank_col"></td>
 +
 +
                    <form method="post"
 +
                          action="/cgi/partsdb/pgroup.cgi?pgroup=iGEM2017&amp;group=SDSZ-China&amp;from=message&amp;isappinstalled=0"
 +
                          enctype="multipart/form-data"></form>
 +
                    <input type="hidden" name="Editing" value="Editing"><input type="hidden" name="part"
 +
                                                                              value="43581"><input type="hidden"
 +
                                                                                                    name="pgroup"
 +
                                                                                                    value="iGEM2017"><input
 +
                        type="hidden" name="group" value="SDSZ-China"></tr>
 +
                <tr>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td class="status_cell cell_white">&nbsp;</td>
 +
                    <td><a class="noul_link part_link"
 +
                          href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2239015">BBa_K2239015</a></td>
 +
                    <td>Coding</td>
 +
                    <td>LDH ( lactate dehydrogenase)</td>
 +
                    <td width="100px">Ruohong Wang</td>
 +
                    <td align="right">1014</td>
 +
                    <td class="blank_col"></td>
 +
                </tr>
 +
                </tbody>
 +
            </table>
 +
 +
            <br>
 +
            <br>
 +
            <br>
 
         </div>
 
         </div>
 
     </div>
 
     </div>

Revision as of 02:12, 2 November 2017


Project

Name Type Description Designer Length
    BBa_K2239001 Generator ChBD-7alphaHSDH Ruohong Wang 1089
    BBa_K2239002 Generator ChBD-7betaHSDH Ruohong Wang 1179
    BBa_K2239003 Generator ChBD-LDH Ruohong Wang 1323
    BBa_K2239004 Generator ChBD-GDH Ruohong Wang 1107
    BBa_K2239005 Generator ChBD-GFP Ruohong Wang 1047
    BBa_K2239006 Generator CBD-7alphaHSDH Ruohong Wang 1080
    BBa_K2239007 Generator CBD-7betaHSDH Ruohong Wang 1170
    BBa_K2239008 Generator CBD-LDH Ruohong Wang 1314
    BBa_K2239009 Generator CBD-GDH Ruohong Wang 1098
    BBa_K2239010 Generator CBD-GFP Ruohong Wang 1038
    BBa_K2239011 Coding 7alpha-HSDH Ruohong Wang 780
    BBa_K2239012 Coding 7beta-HSDH Ruohong Wang 870
    BBa_K2239013 Coding GFP Ruohong Wang 738
    BBa_K2239014 Coding GDH (glucose dehydrogenas) Ruohong Wang 798
    BBa_K2239015 Coding LDH ( lactate dehydrogenase) Ruohong Wang 1014