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

From 2012.igem.org

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           <div><p align="center"><font size="20">Regulation and Control Module</font></p></div>
           <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><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Module"><<< Back to Modules</a></p></div>
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              <h1></h1>
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<h1><p>Overview</p></h1>
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          <p><strong>Overview:</strong><br />
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</div>
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<p>We first introduce a xylose inducible promoter, which can help us control the timing of anti-tumor drug, BMP2 expression and secretion. 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  <i>et al.</i>, 1997) Besides the timing regulation, we 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. By these two regulation systems, our B. hercules can have more reliable and controllable performance.</p>
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<p><strong>Objectives:</strong></p>
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<p>1. To regulate the expression of  BMP2 by a xylose inducible promoter. (Timing regulation.)</p>
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 +
<p>To control the overexpression  of BMP2. (Dosage regulation.)</p>
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</div>
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<div id="paragraph2" class="bodyParagraphs">
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<div align="left">
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<h1><p>Design</p></h1>
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</div>
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  We first introduce a xylose inducible promoter, which can help us control the timing of anti-tumor drug, BMP2 expression and secretion. 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  <i>et al.</i>, 1997) Besides the timing regulation, we 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. By these two regulation systems, our B. hercules can have more reliable and controllable performance.</p>
 
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<p><strong>Objective:</strong></p>
 
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<ol>
 
-
  <li>To regulate the expression of  BMP2 by a xylose inducible promoter. (Timing regulation)</li>
 
-
  <li>To control the overexpression  of BMP2. (Dosage regulation)</li>
 
-
</ol>
 
<p><strong>Our  Module in B. hercules:</strong></p>
<p><strong>Our  Module in B. hercules:</strong></p>
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<ol>
 
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  <li><em>The inducible promoter. <a href="http://partsregistry.org/Part:BBa_K733002">BBa_K733002</a></em></li>
 
-
<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. As a  growth factor, although BMP2 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  <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.<br /><br />
 
-
  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 needed to take into consideration: first, the inducer should not normally exists <em>in  vivo</em>, but can be created in human colon; secondly, the inducer should not vitiate the healthy state of the individual. Besides, although high efficiency of the induction is not strictly required, it will still be considerably helpful if could be achieved.<br /><br />
 
-
  With those concerns in mind,  xylose came across our mind to be the inducer of our B. hercules. Xylose, which is the main building block for hemicellulose, can only be found in plants. Largely absorbed in jejunum before reaching colon, xylose is not present in 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 intestine, and induction can therefore be achieved by xylose delivered in enteric capsule or from anus.<br /><br />
 
-
  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)<br /><br />
 
-
</p>
 
 +
<p>1. <em>The inducible promoter. <a href="http://partsregistry.org/Part:BBa_K733002">BBa_K733002</a></em></p>
 +
<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. As a  growth factor, although BMP2 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  <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.<br />
 +
 +
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 can be created in human colon; b) the inducer should not vitiate the healthy state of the individual. Besides, although high efficiency of the induction is not strictly required, it will still be considerably helpful if could be achieved.<br />
 +
 +
With those concerns in mind, xylose came across our mind to be the inducer of our B. hercules. Xylose, which is the main building block for hemicellulose, can only be found in plants. Largely absorbed in jejunum before reaching colon, xylose is not present in 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 intestine, and induction can therefore be achieved by xylose delivered in enteric capsule or from anus.<br />
 +
 +
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)
 +
</p>
  <p align="center"> <img src="https://static.igem.org/mediawiki/2012/c/ce/Xylose_promoter_1.JPG" width="50%" /></p>
  <p align="center"> <img src="https://static.igem.org/mediawiki/2012/c/ce/Xylose_promoter_1.JPG" width="50%" /></p>
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  <p align="center"> <img src="https://static.igem.org/mediawiki/2012/7/7b/Xylose_promoter_3.JPG" width="60%" /></p>
  <p align="center"> <img src="https://static.igem.org/mediawiki/2012/7/7b/Xylose_promoter_3.JPG" width="60%" /></p>
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  <li><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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<p>2. <em>The Cell Growth Inhibition Device. <a href="http://partsregistry.org/Part:BBa_K733012">BBa_K733012</a></em></li></em></li>
-
<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)<br /><br />
+
-
  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 only  existence 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. <br /><br />
+
-
  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, regarded as "toxin" in this case. On the other  hand, <i>ydcD</i> encodes YdcD (EndoAI), which counteracts the effect of EndoA and regarded as "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 efficient 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></ol>
+
<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)<br />
-
<p align="center">
+
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 only  existence 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. <br />
 +
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, regarded as "toxin" in this case. On the other  hand, <i>ydcD</i> encodes YdcD (EndoAI), which counteracts the effect of EndoA and regarded as "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 efficient 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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 +
<p align="center">
<img src="https://static.igem.org/mediawiki/2012/d/dd/CGIDfunction2..jpg" width="99%" />
<img src="https://static.igem.org/mediawiki/2012/d/dd/CGIDfunction2..jpg" width="99%" />
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<p>Reference:</p>
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  <div id="ftn1">
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<div id="paragraph3" class="bodyParagraphs">
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<div align="left">
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<h1><p>References</p></h1>
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</div>
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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>
<p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Module/Target_binding">Target Binding Module</a></p>
<p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Module/Anti_tumor">Anti-tumor Molecule Secretion Module</a></p>
<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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Revision as of 00:49, 27 September 2012

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

Regulation and Control Module

<<< Back to Modules

Overview

We first introduce a xylose inducible promoter, which can help us control the timing of anti-tumor drug, BMP2 expression and secretion. Our choice of xylose as an inducer stems from its induction efficiency, its little existence and low absorption rate in colon. (Yuasa et al., 1997) Besides the timing regulation, we 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. By these two regulation systems, our B. hercules can have more reliable and controllable performance.

Objectives:

1. To regulate the expression of BMP2 by a xylose inducible promoter. (Timing regulation.)

To control the 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. As a growth factor, although BMP2 triggers the apoptosis of colon cancer cell, it can also stimulate the 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 can be created in human colon; b) the inducer should not vitiate the healthy state of the individual. Besides, 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, xylose came across our mind to be the inducer of our B. hercules. Xylose, which is the main building block for hemicellulose, can only be found in plants. Largely absorbed in jejunum before reaching colon, xylose is not present in 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 intestine, and induction can therefore be achieved by xylose delivered in enteric capsule or from 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 only existence 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, regarded as "toxin" in this case. On the other hand, ydcD encodes YdcD (EndoAI), which counteracts the effect of EndoA and regarded as "anti-toxin" (Pellegrini, O. et al. 2005). By linking ydcE immediately after Bmp2 gene, and put ydcD after Ptms promoter, a relatively low efficient 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