Team:HKUST-Hong Kong/Design Chassis

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<p>Considering the  requirements of our project, <em>Bacillus subtilis</em>, a Gram-positive model organism in lab study, is chosen to be the  chassis in our project. Its ideality can be illustrated in the following  aspects: </p>
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<p>We chose <i>Bacillus subtilis</i> - a model Gram-positive bacterial species often used in laboratory study - as our chassis. It is ideal as a base for designing mechanisms to fulfill our project aims for reasons we mention below.<br>
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<ol>
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  <li>Safety</li>
+
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<p>Safety issues concerning synthetic biology are always challenged, especially when it comes to the area of medical treatment. From bacteria hosts to biowastes, the public has never ceased to raise worries. To ease concerns from the public, researchers try their  best to minimize the biohazardous effect involved in every step of their experiments. Regarding safety issues, we, 2012 HKUST iGEM team, therefore chose to use <em>B. subtilis</em>, a non-pathogenic chassis in our project.</p>
+
<br><b>Safety.</b>
-
<ol>
+
<p>The safety issues concerning synthetic biology are numerous, particularly when it comes to the area of medical treatment. To reduce the chance of recombinant bacteria causing harm, synthetic biologists try to develop and utilize solutions that minimize the potential hazards of their products. Regarding safety issues, we the HKUST iGEM 2012 team chose to use <i>B. subtilis</i>, a non-pathogenic chassis.<br>
-
  <li>Low degree of  virulence:</li>
+
-
<p>Inhabiting gut as a part of normal flora, <em>Bacillus subtilis</em> is considered to be non-pathogenic organism to human. There is few case that human beings are infected by <em>B. subtilis</em>. According to Edberg (Edberg 1991), <em>B. subtilis</em> does not produce significant quantities of extracellular enzymes or possess other virulence factors that would predispose it to cause infection. In other word, <em>B. subtilis</em> possesses low virulence to human and presents low risk of adverse effects to human health.  </p>
+
<br><b>Low degree of virulence.</b>
 +
<p>As a natural member of the human gut microbiome, <i>B. subtilis</i> is considered to be non-pathogenic to humans. There are very few cases of humans being infected by <i>B. subtilis</i>, and of those who have been infected the vast majority had severe immune deficiency. According to Edberg (Edberg 1991), <i>B. subtilis</i> does not produce significant quantities of extracellular enzymes or possess other virulence factors that would predispose it to cause infection. In other word, <i>B. subtilis</i> possesses low virulence to humans and has a low risk of adverse effects to human health. <br>
-
  <li>Establishment of Integration vectors:</li>
+
<br><b>Establishment of integration vectors.</b>
 +
<p>The discovery of natural integration in <i>B. subtilis</i> raised great attention in the field of molecular genetics in 1978. A series of integration vectors for <i>B. subtilis</i> were designed later for different purposes. For our project we employed an integration vector not only for its stability in <i>B. subtilis</i>, but also its advantages in safety. The employment of the integration vector to some extent minimizes the risk of antibiotic resistance spreading within the normal flora in gut. In addition, since the exposure of BMP2 to normal tissues can induce adverse effects, integrating target genes into the genome can reduce the chance of spreading the <i>Bmp2</i> gene through horizontal gene transfer and avoid non-specific drug release in gut.<br>
-
<p align="left">The discovery of natural integration  in <em>Bacillus subtilis</em> raised great  attention in the field of molecular genetics in 1978. Due to its natural  integration, a series of integration vectors for <em>B. subtilis</em> were designed later for different purposes. In our project, we employed an integration vector not only for its stability in <em>B. subtilis</em>, but also its advantages in safety. The employment of integration vector to some extent  minimizes the risk of antibiotic resistance spreading within the normal flora  in gut. In addition, since the exposure of BMP2 to normal tissues can induce adverse effect, integrating target genes into genome can reduce the chance of spreading of <i>Bmp2</i> gene through horizontal gene transfer and avoid non-specific drug release in gut. </p></ol>
+
<br><b>Protein secretion.</b>
 +
<p>Compared with <i>E. coli</i>, another commonly used chassis in iGEM, <i>B. subtilis</i> is preferred because of its reliability in secreting proteins directly to the extracellular environment. As a Gram-positive eubacteria, <i>B. subtilis</i> lacks an outer membrane, featuring only a 10-50nm peptidoglycan layer beyond its the plasma membrane. Since we are aiming for bacterial secretion of BMP2 out into the digestive tract in the vicinity of colon cancer cells, this property suits our purposes well.<br>
-
  <li>Protein secretion:</li>
+
<br><b>Peptide display.</b>
 +
<p>Without an outer membrane, <i>B. subtilis</i> is ideal for displaying items on its surface. The cell wall of <i>B. subtilis</i> is the surface of the bacterium and contains approximately 9% of the total protein in the cell. The discovery and study of cell wall binding devices produced from cell wall bound proteins provides an attractive tool for surface display of peptide (Pooley et al. 1996). Aiming to display the tumor-binding peptide, RPMrel, on bacteria surface, we decided to take advantage of the cell wall display system, LytC, in <i>B. subtilis</i>. Thus, the ease of peptide display serves as one of our reasons to choose <i>B. subtilis</i> as our chassis. <br>
-
<p>Comparing with <em>E. coli</em>, another commonly used  chassis in iGEM, <em>Bacillus subtilis</em> is  preferred because of its high ability in secreting proteins to extracellular  environment. As a Gram-positive eubacteria, <em>B. subtilis</em> lacks an outer membrane, only with a layer of 10 to 50nm  peptidoglycan outside cytoplasmic membrane. Thus <em>Bacillus  subtilis</em> can secrete proteins directly into the extracellular environment more easily. Since  we are aiming for bacterial secretion of BMP2 out to the colon, this property suits our purposes well.  </p>
+
<br><b>Part of gut commensal flora.</b>
 +
<p><i>B. subtilis</i> does not only exist widely in nature, but also contributes to part of the normal flora in the gut (Collin and Gibson 1999). Regarded as a probiotic, it has been proved to have positive effects on patients suffering from functional abdominal bloating (Corazza et al. 1992). Using <i>B. subtilis</i> as our chassis will in theory mean we will not be introducing any exotic species into the gut. <br>
-
  <li>Peptide  displaying:</li>
+
<br><b>References.</b><br>
 +
<p>Collins M. D. and Gibson G. R.. 1999. Probiotics, prebiotics and synbiotics: Approaches for modulating the microbial ecology of the gut. <i>Am. J. Clin. Nutr.</i> 69:1052S–1057S

-
<p>Without the outer membrane, <em>Bacillus subtilis</em> is ideal for peptide displaying. The cell wall of <em>B. subtilis</em> is the surface of the bacterium and contains  approximately 9% of the total protein. The discovery and study in cell wall binding modules from cell wall bound proteins provides an attractive tool  for surface display of peptide. (Pooley et al. 1996) Aiming to display the  tumor-binding peptide, RPMrel, on bacteria surface, we decided to take advantage of the cell wall displaying system, LytC, in <em>Bacillus subtilis</em>. Thus, the ease of peptide displaying serves as one of our reasons to choose <em>B. subtilis</em> as our chassis. </p>
+
<p>Corazza G.R., Benati G., Strocchi A., Sorge M. & Gasbarrini G.. 1992. Treatment with <i>Bacillus subtilis</i> reduces intestinal hydrogen production in patients with gaseous symptoms. <i>Current therapeutic research</i>. 52.1: 144-151
-
  <li>Normal flora in  gut:</li>
+
<p>Edberg, S.C.. 1991. US EPA human health assessment: <i>Bacillus subtilis</i>. Unpublished, U.S. Environmental Protection Agency, Washington, D.C.

-
<p><em>B.  subtilis</em> not only exists widely in the nature, but also contributes to part of the normal flora  in gut. (Collin and Gibson 1999) Regarded as a probiotic, it has been proved to have positive effects on patients suffering from gaseous symptom  (Corazza et al. 1992). Using <em>B.  subtilis</em> as our chassis, in theory, will not introduce any exotic species into gut either. </p></ol>
+
<p>Pooley H. M., Merchante R. and Karamata D.. 1996. Overall protein content and induced enzyme components of the periplasm of <i>Bacillus subtilis</i>. <i>Microb. Drug Resist</i>.2:9–15.</p>
-
<p><strong>References: </strong><br />
+
 
-
  Edberg,S.C.. 1991. US EPA human health assessment: <i>Bacillus subtilis</i>. Unpublished, U.S.  Environmental Protection Agency, Washington, D.C.<br />
+
</div>
-
  Pooley H. M., Merchante R. and Karamata D.. 1996. Overall protein content and induced enzyme components of the periplasm of <em>Bacillus subtilis</em>. <em>Microb. Drug Resist</em>.2:9–15.<br />
+
-
  Collins M. D. and Gibson G. R.. 1999. Probiotics, prebioticsand  synbiotics: Approaches for modulating the microbial ecology of the gut. <i>Am. J.  Clin. Nutr.</i> 69:1052S–1057S<br />
+
-
  Corazza G.R., Benati G., Strocchi A., Sorge M. &amp; Gasbarrini G.. 1992. Treatment  with <i>Bacillus subtilis</i> reduces intestinal hydrogen production in patients with  gaseous symptoms. <em>Current therapeutic  research. </em>52.1: 144-151</p>
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Latest revision as of 19:07, 26 September 2012

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

Design - Chassis

We chose Bacillus subtilis - a model Gram-positive bacterial species often used in laboratory study - as our chassis. It is ideal as a base for designing mechanisms to fulfill our project aims for reasons we mention below.

Safety.

The safety issues concerning synthetic biology are numerous, particularly when it comes to the area of medical treatment. To reduce the chance of recombinant bacteria causing harm, synthetic biologists try to develop and utilize solutions that minimize the potential hazards of their products. Regarding safety issues, we the HKUST iGEM 2012 team chose to use B. subtilis, a non-pathogenic chassis.

Low degree of virulence.

As a natural member of the human gut microbiome, B. subtilis is considered to be non-pathogenic to humans. There are very few cases of humans being infected by B. subtilis, and of those who have been infected the vast majority had severe immune deficiency. According to Edberg (Edberg 1991), B. subtilis does not produce significant quantities of extracellular enzymes or possess other virulence factors that would predispose it to cause infection. In other word, B. subtilis possesses low virulence to humans and has a low risk of adverse effects to human health.

Establishment of integration vectors.

The discovery of natural integration in B. subtilis raised great attention in the field of molecular genetics in 1978. A series of integration vectors for B. subtilis were designed later for different purposes. For our project we employed an integration vector not only for its stability in B. subtilis, but also its advantages in safety. The employment of the integration vector to some extent minimizes the risk of antibiotic resistance spreading within the normal flora in gut. In addition, since the exposure of BMP2 to normal tissues can induce adverse effects, integrating target genes into the genome can reduce the chance of spreading the Bmp2 gene through horizontal gene transfer and avoid non-specific drug release in gut.

Protein secretion.

Compared with E. coli, another commonly used chassis in iGEM, B. subtilis is preferred because of its reliability in secreting proteins directly to the extracellular environment. As a Gram-positive eubacteria, B. subtilis lacks an outer membrane, featuring only a 10-50nm peptidoglycan layer beyond its the plasma membrane. Since we are aiming for bacterial secretion of BMP2 out into the digestive tract in the vicinity of colon cancer cells, this property suits our purposes well.

Peptide display.

Without an outer membrane, B. subtilis is ideal for displaying items on its surface. The cell wall of B. subtilis is the surface of the bacterium and contains approximately 9% of the total protein in the cell. The discovery and study of cell wall binding devices produced from cell wall bound proteins provides an attractive tool for surface display of peptide (Pooley et al. 1996). Aiming to display the tumor-binding peptide, RPMrel, on bacteria surface, we decided to take advantage of the cell wall display system, LytC, in B. subtilis. Thus, the ease of peptide display serves as one of our reasons to choose B. subtilis as our chassis.

Part of gut commensal flora.

B. subtilis does not only exist widely in nature, but also contributes to part of the normal flora in the gut (Collin and Gibson 1999). Regarded as a probiotic, it has been proved to have positive effects on patients suffering from functional abdominal bloating (Corazza et al. 1992). Using B. subtilis as our chassis will in theory mean we will not be introducing any exotic species into the gut.

References.

Collins M. D. and Gibson G. R.. 1999. Probiotics, prebiotics and synbiotics: Approaches for modulating the microbial ecology of the gut. Am. J. Clin. Nutr. 69:1052S–1057S


Corazza G.R., Benati G., Strocchi A., Sorge M. & Gasbarrini G.. 1992. Treatment with Bacillus subtilis reduces intestinal hydrogen production in patients with gaseous symptoms. Current therapeutic research. 52.1: 144-151

Edberg, S.C.. 1991. US EPA human health assessment: Bacillus subtilis. Unpublished, U.S. Environmental Protection Agency, Washington, D.C.


Pooley H. M., Merchante R. and Karamata D.. 1996. Overall protein content and induced enzyme components of the periplasm of Bacillus subtilis. Microb. Drug Resist.2:9–15.