Team:HKUST-Hong Kong/Module/Regulation and control

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               <div class="Content_Buttons"><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Members">Members</a></p></div>
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               <div class="Content_Buttons"><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Project_Abstraction">Project Abstract</a></p></div>
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               <div class="Content_Buttons"><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Characterization">Characterization</a></p></div>
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               <div class="Content_Buttons"><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Achievement">Achievement</a></p></div>
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                <div class="Content_Buttons"><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Medal_Requirements">Medal Requirements</a></p></div>
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                <div class="Content_Buttons""><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Acknowledgement">Acknowledgement</a></p></div>
               <div class="Content_Buttons"><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Glossary">Glossary</a></p></div>
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           <div><p align="center"><font size="20">REGULATION AND CONTROL MODULE</font></p></div>
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<div><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Module"><<< Back to Module</a></p></div>
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           <div><p align="center"><font size="20">Regulation and Control Module</font></p></div>
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<div><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Module"><<< Back to Modules</a></p></div>
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          <p><strong>Overview:</strong><br />
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  Our  module aims at regulating our synthetic bacteria <em>B. hercules. </em>We first introduce a xylose inducible promoter, which  can help us control the timing of BMP-2 expression. Our choice of xylose as an inducer stems from its induction efficiency, its little existence and low absorption rate in colon.<a href="#_ftn1" name="_ftnref1" title="" id="_ftnref1"> </a>(Yuasa  et al., 1997) Besides the timing regulation, we introduce a cell growth inhibition device to prevent the overexpression of BMP-2. This device is achieved by a balance between a toxin and antitoxin pair ydcE and ydcD. By these two regulation systems, our <em>B. hercules</em> can produce more reliable and controllable performance.</p>
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<h1><p>Overview</p></h1>
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<p><strong>Objective:</strong></p>
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<ol>
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  <li>To regulate the expression of BMP-2 by a xylose inducible promoter. (Timing regulation)</li>
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<p>We first introduced a xylose inducible promoter that, when used to express the anti-tumor drug BMP2, can help us control the timing of its expression and secretion. We chose xylose as an inducer because of its induction efficiency, relative scarcity and low absorption rate in the colon.<a href="#_ftn1" name="_ftnref1" title="" id="_ftnref1"> </a>(Yuasa  <i>et al.</i>, 1997) Besides the timing regulation, we further introduce a cell growth inhibition device to prevent the overexpression of BMP2. This device is achieved by a balance between a toxin and antitoxin pair, YdcE and YdcD. Under these two regulation systems, our B. hercules can have more reliable and controllable performance.</p>
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  <li>To control the overexpression of BMP-2. (Dosage regulation)</li>
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<p><strong>Objectives:</strong></p>
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<p><strong>Our Module in <em>B. hercules:</em></strong></p>
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<ol>
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<p>1. To provide external control over the induction of BMP2 expression, by using a xylose inducible promoter. (Timing regulation.)</p>
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  <li><em>The inducible promoter.</em></li>
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<p>In the consideration of our <em>B. hercules</em>, one of our concerns is that our bacteria secrete BMP-2 before its binding with colon cancer cells. As a  growth factor, while BMP-2 triggers the apoptosis of colon cancer cell, it can  also stimulate the proliferation of normal epithelial cells in digestive tract. <a href="#_ftn2" name="_ftnref2" title="" id="_ftnref2"> </a>(Zhang  et al., 2012) Thus, we intend to introduce a timing regulatory system into our <em>B. hercules</em>, which can be achieved by an inducible promoter.<br /><br />
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<p>To prevent overexpression of BMP2. (Dosage regulation.)</p>
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  Admittedly, there are various kinds  of induction systems in <em>Bacillus subtilis</em>. However, to achieve the induction when our <em>B. hercules</em> is inside human colon, two conditions are required: first, the condition should not occur normally <em>in  vivo</em>, but can be created in human colon; secondly, the inducer should not vitiate the healthy state of an individual. Besides, the efficiency of the induction is not required, but will be considerably helpful.<br /><br />
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  With those questions in mindxylose is chosen as our inducer for <em>B. hercules.</em> It is the main building block for hemicellulose, which can only be found in plant. Largely absorbed in jejunum before reaching colon, xylose is not present in colon.1(Yuasa et al., 1997) Besides, the absorption rate of xylose in colon is low. Thus, well scheduled diet and medication can prevent the interaction of xylose and <em>B. hercules</em> before intestine and induction can therefore achieved by xylose delivered in enteric capsule or from anus. . <br /><br />
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  Besides its rare existence in the  human colon, xylose is an efficient inducer as for PxylA promoter. When ligated  with gene <em>bgaB</em>, 200-fold induction  was achieved 30 minutes after the induction of xylose.<a href="#_ftn3" name="_ftnref3" title="" id="_ftnref3"> </a> (Kim, L. et al. 1996)<br /><br />
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<h1><p>Design</p></h1>
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<p><strong>Our Module in B. hercules:</strong></p>
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<p>1. <em>The inducible promoter. <a href="http://partsregistry.org/Part:BBa_K733002">BBa_K733002</a></em></p>
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<p>In the consideration of our B. hercules, one of our concerns is that our bacteria may secrete BMP2 before its binding to colon cancer cells. Although BMP2 triggers the apoptosis of colon cancer cell, its better known function is the stimulate of growth and proliferation of normal epithelial cells in digestive tract. <a href="#_ftn2" name="_ftnref2" title="" id="_ftnref2"> </a>(Zhang  <i>et al.</i>, 2012) Thus, we intend to introduce a regulatory timing system into our B. hercules by incorporating an inducible promoter into our device.</p>
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<p>Admittedly, there are many different induction systems in <em>Bacillus subtilis</em>. However, to achieve the induction when our B. hercules is inside human colon, two conditions need to be taken into consideration: a) the inducer should not normally exist <em>in  vivo</em>, but the option should be there to make it available in the human colon; b) the inducer should not vitiate the healthy state of the individual. Furthermore, although high efficiency of the induction is not strictly required, it will still be considerably helpful if could be achieved.</p>
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<p>With those concerns in mind we decided on xylose to induce our B. hercules. Xylose, which is the main building block for hemicellulose, can only be found in plants. Largely absorbed in the jejunum before reaching colon, xylose is not typically present in the colon.(Yuasa <i>et al.</i>, 1997) Besides, the absorption rate of xylose in colon is low indicated by Yuasa. Thus, well scheduled diet and medication can prevent the interaction of xylose and B. hercules in an earlier stage of the digestive tract, and induction can therefore be achieved by xylose delivered in enteric capsules or from the anus.</p>
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<p>Besides its rare existence in the  human colon, xylose is an efficient inducer as for <i>PxylA</i> promoter. When ligated  with gene <em>bgaB</em>, 200-fold induction  was achieved 30 minutes after the induction of xylose.<a href="#_ftn3" name="_ftnref3" title="" id="_ftnref3"> </a> (Kim <i>et al</i>. 1996)
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  <li><em>The Cell Growth Inhibition Device.</em></li>
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<p>  Considering the unexpected proliferation of normal colon cells caused by over-dose BMP-2, a regulatory system is necessary for the dosage  control of BMP-2 expression.2 (Zhang et  al., 2012)<br /><br />
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<p align="center"> <img src="https://static.igem.org/mediawiki/2012/c/ce/Xylose_promoter_1.JPG" width="50%" /></p>
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  In order to build  this system, we came up with a cell growth inhibition device to regulate. Understanding that toxin-antitoxin operons exist abundantly in bacteria, we intend to link the expression of BMP-2 with a toxin gene. However, the only  existence of the toxin gene is not enough. Stabilization, to a certain extent, is necessary, so that our <em>B. hercules</em> will not die after a low level of BMP-2 expression.<br /><br />
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<p align="center"> <img src="https://static.igem.org/mediawiki/2012/e/eb/Xylose_promoter_2.JPG" width="60%" /></p>
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  In order to implement the ideas above, a toxin-antitoxin pair – ydcE and ydcD – is used.  ydcE can encode an endoribonuclease – EndoA, which can cause cell growth inhibition. (For specific information, please click<u> here</u>) On the other  hand, ydcD can encode YdcD, counteracting the effect of EndoA. <a href="#_ftn4" name="_ftnref4" title="" id="_ftnref4"> </a>(Pellegrini, O. et al. 2005) By linking ydcE immediately after BMP-2 gene, and put ydcD after  pTms promoter, a relatively low efficient constitutive promoter, EndoA can be  expressed simultaneously with the expression of BMP-2 under the control of  xylose inducible promoter, and cell growth inhibition will not occur until the  produced EndoA outweighs the effect of accumulated YdcD.</p></ol>
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<p align="center"> <img src="https://static.igem.org/mediawiki/2012/7/7b/Xylose_promoter_3.JPG" width="60%" /></p>
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<div><br><br>
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<p>Reference:</p>
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<p>2. <em>The Cell Growth Inhibition Device. <a href="http://partsregistry.org/Part:BBa_K733012">BBa_K733012</a></em></li></em></li>
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  <div id="ftn1">
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<p>Considering the problems caused by the unexpected proliferation of normal colon cells induced by over-dose BMP2, a regulatory system is necessary for the dosage  control of BMP2 expression.(Zhang <i>et  al.</i>, 2012)</p>
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<p>In order to build  this controlling system, we came up with a cell growth inhibition device to manage this task. Understanding that toxin-antitoxin operons exist abundantly in bacteria, we intend to link the expression of BMP2 with a toxin gene. However, the lone presence of the toxin gene is not enough. Stabilization, to a certain extent, is necessary, so that our B. hercules will not die after a low level of BMP2 expression. And this short-term stabilization could be achieved by introducing the corresponding anti-toxin gene of the previous toxin gene. </p>
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<p>In order to practically implement the ideas above, a toxin-antitoxin pair – YdcE and YdcD – is used.  <i>ydcE</i> encodes an endoribonuclease – EndoA, which causes cell growth inhibition, and is regarded as the "toxin" in this case. On the other  hand, <i>ydcD</i> encodes YdcD (EndoAI), which counteracts the effect of EndoA and is regarded as the "anti-toxin" <a href="#_ftn4" name="_ftnref4" title="" id="_ftnref4"> </a>(Pellegrini, O. et al. 2005). By linking <i>ydcE</i> immediately after <i>Bmp2</i> gene, and put <i>ydcD</i> after  <i>Ptms</i> promoter, a relatively low efficiency constitutive promoter, EndoA can be  expressed simultaneously with the expression of BMP2 under the control of  xylose inducible promoter, and cell growth inhibition will not occur until the  produced EndoA outweighs the effect of accumulated YdcD (EndoAI).</p>
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<img src="https://static.igem.org/mediawiki/2012/d/dd/CGIDfunction2..jpg" width="99%" />
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<h1><p>References</p></h1>
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<div id="ftn1">
     <a href="#_ftnref1" name="_ftn1" title="" id="_ftn1"> </a> Yuasa, H., Kuno, C., &amp; Watanabe, J. (1997).  Comparative assessment of D-xylose absorption between small intestine and large  intestine..&nbsp;<em>The journal of  pharmacy and pharmocology</em>,<em>49</em>, 26-29. </div>
     <a href="#_ftnref1" name="_ftn1" title="" id="_ftn1"> </a> Yuasa, H., Kuno, C., &amp; Watanabe, J. (1997).  Comparative assessment of D-xylose absorption between small intestine and large  intestine..&nbsp;<em>The journal of  pharmacy and pharmocology</em>,<em>49</em>, 26-29. </div>
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<p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Module/Target_binding">Target Binding Module</a></p>
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<p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Module/Anti_tumor">Anti-tumor Molecule Secretion Module</a></p>
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<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Introduction">Introduction</a></p></li>
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<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Supervisor">Supervisor</a></p></li>
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</ol>
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</div>
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<div class="Sitemap_Content">
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<li><p><b>Project</b></p><ol>
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<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Project_Abstraction">Abstract</a></p></li>
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<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Background_and_Motive">Motive</a></p></li>
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<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Design_Overview">Design - Overview</a></p></li>
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<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Design_Module">Design - Module</a></p></li>
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<p>-- <a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Module/Target_binding">Target Binding Module</a></p>
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<p>-- <a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Module/Anti_tumor">Anti-tumor Molecule Secretion Module</a></p>
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<p>-- <a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Module/Regulation_and_control">Regulation and Control Module</a></p>
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<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Design_Chassis">Design - Chassis</a></p></li></ol>
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</div>
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<div class="Sitemap_Content">
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<li><p><b>Wet Lab</b></p><ol>
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<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Parts_and_Device">Parts and Devices</a></p></li>
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<p>-- <a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Parts_and_Device">Overview</a></p>
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<p>-- <a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Construction">Construction</a></p>
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<p>-- <a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Assembly">Assembly</a></p>
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<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Notebook">Notebook</a></p></li>
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<p>-- <a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Notebook/Logbook">Logbook</a></p>
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<p>-- <a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Notebook/Protocol">Protocol</a></p>
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<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Characterization">Characterization</a></p></li>
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<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Achievement">Achievement</a></p></li>
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<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Future_Work">Future Work</a></p></li></ol>
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</div>
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<div class="Sitemap_Content">
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<li><p><b>Human Practice</b></p><ol>
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<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Overview">Overview</a></p></li>
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<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Interview">Interview</a></p></li>
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<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Presentation">Presentation</a></p></li>
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<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Calendar">Calendar</a></p></li></ol>
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</div>
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<div class="Sitemap_Content">
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<li><p><b>Extras</b></p><ol>
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<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Medal_Requirements">Medal Requirements</a></p></li>
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<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Safety">Safety</a></p></li>
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<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Attribution">Attribution</a></p></li>
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<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Acknowledgement">Acknowledgement</a></p></li>
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<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Glossary">Glossary</a></p></li></ol>
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</div>
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</div>
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Latest revision as of 14:14, 21 October 2012

Team:HKUST-Hong Kong - 2012.igem.org

Regulation and Control Module

<<< Back to Modules

Overview

We first introduced a xylose inducible promoter that, when used to express the anti-tumor drug BMP2, can help us control the timing of its expression and secretion. We chose xylose as an inducer because of its induction efficiency, relative scarcity and low absorption rate in the colon. (Yuasa et al., 1997) Besides the timing regulation, we further introduce a cell growth inhibition device to prevent the overexpression of BMP2. This device is achieved by a balance between a toxin and antitoxin pair, YdcE and YdcD. Under these two regulation systems, our B. hercules can have more reliable and controllable performance.

Objectives:

1. To provide external control over the induction of BMP2 expression, by using a xylose inducible promoter. (Timing regulation.)

To prevent overexpression of BMP2. (Dosage regulation.)

Design

Our Module in B. hercules:

1. The inducible promoter. BBa_K733002

In the consideration of our B. hercules, one of our concerns is that our bacteria may secrete BMP2 before its binding to colon cancer cells. Although BMP2 triggers the apoptosis of colon cancer cell, its better known function is the stimulate of growth and proliferation of normal epithelial cells in digestive tract. (Zhang et al., 2012) Thus, we intend to introduce a regulatory timing system into our B. hercules by incorporating an inducible promoter into our device.

Admittedly, there are many different induction systems in Bacillus subtilis. However, to achieve the induction when our B. hercules is inside human colon, two conditions need to be taken into consideration: a) the inducer should not normally exist in vivo, but the option should be there to make it available in the human colon; b) the inducer should not vitiate the healthy state of the individual. Furthermore, although high efficiency of the induction is not strictly required, it will still be considerably helpful if could be achieved.

With those concerns in mind we decided on xylose to induce our B. hercules. Xylose, which is the main building block for hemicellulose, can only be found in plants. Largely absorbed in the jejunum before reaching colon, xylose is not typically present in the colon.(Yuasa et al., 1997) Besides, the absorption rate of xylose in colon is low indicated by Yuasa. Thus, well scheduled diet and medication can prevent the interaction of xylose and B. hercules in an earlier stage of the digestive tract, and induction can therefore be achieved by xylose delivered in enteric capsules or from the anus.

Besides its rare existence in the human colon, xylose is an efficient inducer as for PxylA promoter. When ligated with gene bgaB, 200-fold induction was achieved 30 minutes after the induction of xylose. (Kim et al. 1996)

2. The Cell Growth Inhibition Device. BBa_K733012

Considering the problems caused by the unexpected proliferation of normal colon cells induced by over-dose BMP2, a regulatory system is necessary for the dosage control of BMP2 expression.(Zhang et al., 2012)

In order to build this controlling system, we came up with a cell growth inhibition device to manage this task. Understanding that toxin-antitoxin operons exist abundantly in bacteria, we intend to link the expression of BMP2 with a toxin gene. However, the lone presence of the toxin gene is not enough. Stabilization, to a certain extent, is necessary, so that our B. hercules will not die after a low level of BMP2 expression. And this short-term stabilization could be achieved by introducing the corresponding anti-toxin gene of the previous toxin gene.

In order to practically implement the ideas above, a toxin-antitoxin pair – YdcE and YdcD – is used. ydcE encodes an endoribonuclease – EndoA, which causes cell growth inhibition, and is regarded as the "toxin" in this case. On the other hand, ydcD encodes YdcD (EndoAI), which counteracts the effect of EndoA and is regarded as the "anti-toxin" (Pellegrini, O. et al. 2005). By linking ydcE immediately after Bmp2 gene, and put ydcD after Ptms promoter, a relatively low efficiency constitutive promoter, EndoA can be expressed simultaneously with the expression of BMP2 under the control of xylose inducible promoter, and cell growth inhibition will not occur until the produced EndoA outweighs the effect of accumulated YdcD (EndoAI).

References

Yuasa, H., Kuno, C., & Watanabe, J. (1997). Comparative assessment of D-xylose absorption between small intestine and large intestine.. The journal of pharmacy and pharmocology,49, 26-29.

Zhang J, Ge Y, Sun L, Cao J, Wu Q, Guo L, Wang Z. Effect of Bone Morphogenetic Protein-2 on Proliferation and Apoptosis of Gastric Cancer Cells. Int J Med Sci 2012; 9(2):184-192.

Kim, L., Mogk, A., & Schumann, W. (1996). A xylose-inducible Bacillus subtilis integration vector and its application. Gene181(1-2), 71-76.

Pellegrini, O., Mathy, N., Gogos, A., Shapiro, L., & Condon, C. (2005). The Bacillus subtilis ydcDE operon encodes an endoribonuclease of the MazFPemK family and its inhibitor.Molecular Microbiology56(5), 1139-1148.

Target Binding Module

Anti-tumor Molecule Secretion Module