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

From 2012.igem.org

(Difference between revisions)
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           </div>
           </div>
           <p><strong>Overview:</strong><br />
           <p><strong>Overview:</strong><br />
-
   Our  module aims at regulating our synthetic bacteria B. hercules. We first introduce a xylose inducible promoter, which can help us control the timing of BMP2 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 BMP2. 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>
+
   Our  module aims at regulating our synthetic bacteria B. hercules. We first introduce a xylose inducible promoter, which can help us control the timing of BMP2 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  <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 <em>B. hercules</em> can produce more reliable and controllable performance.</p>
<p><strong>Objective:</strong></p>
<p><strong>Objective:</strong></p>
<ol>
<ol>
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   <li>To control the overexpression  of BMP2. (Dosage regulation)</li>
   <li>To control the overexpression  of BMP2. (Dosage regulation)</li>
</ol>
</ol>
-
<p><strong>Our  Module in <em>B. hercules:</em></strong></p>
+
<p><strong>Our  Module in B. hercules:</strong></p>
<ol>
<ol>
   <li><em>The inducible promoter. <a href="http://partsregistry.org/Part:BBa_K733002">BBa_K733002</a></em></li>
   <li><em>The inducible promoter. <a href="http://partsregistry.org/Part:BBa_K733002">BBa_K733002</a></em></li>
-
<p>In the consideration of our <em>B. hercules</em>, one of our concerns is that  our bacteria secrete BMP2 before its binding with colon cancer cells. As a  growth factor, while 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  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 />
+
<p>In the consideration of our B. hercules, one of our concerns is that  our bacteria secrete BMP2 before its binding with colon cancer cells. As a  growth factor, while 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  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 />
-
   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 />
+
   Admittedly, there are various kinds  of induction systems in <em>Bacillus subtilis</em>.  However, to achieve the induction when our B.  hercules 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 />
-
   With those questions in mind,  xylose 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.(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 />
+
   With those questions in mind,  xylose is chosen as our inducer for B. hercules. 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.(Yuasa <i>et al.</i>, 1997) Besides,  the absorption rate of xylose in colon is low. Thus, well scheduled diet and  medication can prevent the interaction of xylose and B. hercules before intestine and induction can therefore 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 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 />
   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 />
</p>
</p>
   <li><em>The Cell Growth Inhibition Device. <a href="http://partsregistry.org/Part:BBa_K733012">BBa_K733012</a></em></li></em></li>
   <li><em>The Cell Growth Inhibition Device. <a href="http://partsregistry.org/Part:BBa_K733012">BBa_K733012</a></em></li></em></li>
-
<p>Considering the unexpected  proliferation of normal colon cells caused by over-dose BMP2, a regulatory system is necessary for the dosage  control of BMP2 expression.(Zhang et  al., 2012)<br /><br />
+
<p>Considering the unexpected  proliferation of normal colon cells caused 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 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 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 <em>B. hercules</em> will not die after a low level of BMP2 expression.<br /><br />
   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 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 <em>B. hercules</em> will not die after a low level of BMP2 expression.<br /><br />
   In order to  implement the ideas above, a toxin-antitoxin pair – ydcE and ydcD – is used.  <i>ydcE</i> can encode an endoribonuclease – EndoA, which can cause cell growth  inhibition. On the other  hand, <i>ydcD</i> can encode YdcD, counteracting the effect of EndoA. <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.</p></ol>
   In order to  implement the ideas above, a toxin-antitoxin pair – ydcE and ydcD – is used.  <i>ydcE</i> can encode an endoribonuclease – EndoA, which can cause cell growth  inhibition. On the other  hand, <i>ydcD</i> can encode YdcD, counteracting the effect of EndoA. <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.</p></ol>

Revision as of 15:50, 26 September 2012

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

Regulation and Control Module

<<< Back to Module

Overview:
Our module aims at regulating our synthetic bacteria B. hercules. We first introduce a xylose inducible promoter, which can help us control the timing of BMP2 expression. 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 produce more reliable and controllable performance.

Objective:

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

Our Module in B. hercules:

  1. The inducible promoter. BBa_K733002
  2. In the consideration of our B. hercules, one of our concerns is that our bacteria secrete BMP2 before its binding with colon cancer cells. As a growth factor, while 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 timing regulatory system into our B. hercules, which can be achieved by an inducible promoter.

    Admittedly, there are various kinds of induction systems in Bacillus subtilis. However, to achieve the induction when our B. hercules is inside human colon, two conditions are required: first, the condition should not occur normally in vivo, 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.

    With those questions in mind, xylose is chosen as our inducer for B. hercules. 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.(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 B. hercules before intestine and induction can therefore 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, L. et al. 1996)

  3. The Cell Growth Inhibition Device. BBa_K733012
  4. Considering the unexpected proliferation of normal colon cells caused 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 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 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.

    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. On the other hand, ydcD can encode YdcD, counteracting the effect of EndoA. (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.



Reference:

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