Team:HKUST-Hong Kong/Module/Target binding

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.Upper_Logos#HKUST_Logo{
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<body>
<body>
         <div id="Navigation_top">
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           <a href="https://2012.igem.org/Main_Page"><img id="iGEM_Logo" class="Upper_Logos" src="https://static.igem.org/mediawiki/2012/3/32/Igem-logo.png"></a>
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           <a href="http://www.ust.hk"><img id="HKUST_Logo" class="Upper_Logos" src=""></a>
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           <div class="Navigation_Buttons" id="Team" align="center"><h3><p>TEAM</p></h3></div>
           <div class="Navigation_Buttons" id="Team" align="center"><h3><p>TEAM</p></h3></div>
           <div class="Navigation_Buttons" id="Project" align="center"><h3><p>PROJECT</p></h3></div>
           <div class="Navigation_Buttons" id="Project" align="center"><h3><p>PROJECT</p></h3></div>
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               <div class="Content_Buttons"><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Members">Members</a></p></div>
               <div class="Content_Buttons"><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Members">Members</a></p></div>
               <div class="Content_Buttons"><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Advisors">Advisors</a></p></div>
               <div class="Content_Buttons"><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Advisors">Advisors</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>
 
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           <div class="Navigation_Content" id="Project_Content">
           <div class="Navigation_Content" id="Project_Content">
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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/Project_Abstraction">Abstract</a></p></div>
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               <div class="Content_Buttons"><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Background_and_Motive">Background and<br> Motive</a></p></div>
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               <div class="Content_Buttons"><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Background_and_Motive">Motive</a></p></div>
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               <div class="Content_Buttons"><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Project">Project Description</a></p></div>
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               <div class="Content_Buttons"><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Design_Overview">Design - Overview</a></p></div>
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              <div class="Content_Buttons"><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Module">Module</a></p></div>
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               <div class="Content_Buttons"><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Design_Module">Design - Module</a></p></div>
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               <div class="Content_Buttons"><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Chassis">Chassis</a></p></div>
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               <div class="Content_Buttons"><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Design_Chassis">Design - Chassis</a></p></div>
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              <div class="Content_Buttons"><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Safety">Safety</a></p></div>
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               <div class="Content_Buttons"><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Expectation">Expectation</a></p></div>
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              <div class="Content_Buttons"><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Potential_Application">Potential applicaton</a></p></div>
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           </div>
           <div class="Navigation_Content" id="Wet_lab_Content" align="center">
           <div class="Navigation_Content" id="Wet_lab_Content" align="center">
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               <div class="Content_Buttons"><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Characterization">Characterization</a></p></div>
               <div class="Content_Buttons"><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Characterization">Characterization</a></p></div>
               <div class="Content_Buttons"><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Achievement">Achievement</a></p></div>
               <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/Prospect">Prospect</a></p></div>
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               <div class="Content_Buttons"><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Future_Work">Future Work</a></p></div>
           </div>
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           <div class="Navigation_Content" id="Human_practice_Content" align="center">
           <div class="Navigation_Content" id="Human_practice_Content" align="center">
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           </div>
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           <div class="Navigation_Content" id="Extras_Content" align="center">
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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/Safety">Safety</a></p></div>
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                <div class="Content_Buttons""><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Attribution">Attribution</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>
               <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 class="Content_Buttons"><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Site_Map">Site Map</a></p></div>
 
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              <div class="Content_Buttons"><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Photo_Gallery">Photo Gallery</a></p></div>
 
           </div>
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         </div>
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           <div><p align="center"><font size="20">TARGET BINDING 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 id="paragraph1" class="bodyParagraphs">
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          <div align="left">
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           <div><p align="center"><font size="20">Target Binding Module</font></p></div>
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              <h1>Objective</h1>
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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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          </div>
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          <p>Our decision to pursue colorectal carcinoma suppression arose from two key points obtained from preliminary  research: 1) bone morphogenetic protein 2 (BMP-2) suppresses the growth of colon cancer cell growth <em>in vivo</em>, and 2) the phage display peptide  RPMrel confers specific and preferential binding to non-differentiated colon cancer cells. </p>
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<p>With these two pieces of knowledge we had respectively: 1) our carcinoma suppression drug, and 2) a tool for  specifically targeting cancerous cells. Thus the objective of this module was to identify and then construct a suitable mechanism making use of the RPMrel peptide to make the delivery of BMP-2 targeted.  </p>
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<div id="paragraph1" class="bodyParagraphs">
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<p>&nbsp;</p>
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<div align="left">
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<p><strong><u>DESIGN</u></strong></p>
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<h1><p>Overview</p></h1>
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<p><strong>Considering limitations.</strong></p>
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</div>
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<p>Design of a solution starts with considering existing limitations. Since this is iGEM, the clearest limitation was that the solution must be a biological one and thus must involve a living component. Only a certain set of living organisms lie within our reasonable capacity to engineer them, and of these we decided on <em>Bacillus subtilis</em> (see Chassis page). </p>
+
 
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<p>We then examined the treatment environment. Carcinomas of the colon protrude into the digestive tract and items within the tract can interact with them directly. We decided on the concept that our biological system would be ingested, then produce and release the drug in the vicinity of the tumor for direct action. </p>
+
<p>Our decision to pursue colorectal carcinoma suppression arose from two key points obtained from preliminary  research: 1) bone morphogenetic protein 2 (BMP2) suppresses the growth of colon cancer cell growth <em>in vivo</em>, and 2) the phage display peptide  RPMrel confers specific and preferential binding to non-differentiated colon cancer cells. </p>
-
<p>To do this, RPMrel had to be expressed on the cell surface in a functional form. We conducted research into several methods to do this on <em>B. subtilis</em> and concluded that cell wall expression of the peptide was ideal. Imperial College London&rsquo;s 2010 team had performed that same task using the cell wall binding domain of the hydrolase lytC as their peptide anchor and a helical linker of their design. We decided to the employ that same system for surface expression of RPMrel. </p>
+
 
-
          </div>
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<p>With these two pieces of knowledge we have respectively: 1) our carcinoma suppression drug, and 2) a tool for  specifically targeting cancerous cells. Thus the objective of this module was to identify and then construct a suitable mechanism making use of the RPMrel peptide to target the delivery of BMP2. </p>
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  <div id="paragraph2" class="bodyParagraphs">
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          <div align="left">
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</div>
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              <h1>Design</h1>
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          </div>
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<div id="paragraph2" class="bodyParagraphs">
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          <p>Using phage display to compile peptide libraries that confer specific binding to certain antigens is a now common way to come up with useful peptides. RPMrel, a 9 amino acid disulfide-constrained peptide, was screened out of the New England Biolabs <a href="http://www.neb.com/nebecomm/products/producte8100.asp">Ph.D.-C7C library</a> for positive binding  to poorly differentiated HT-29 cells, and negative binding to well  differentiated HCT-116 cells. All peptides in the Ph.D.-C7C library have random  sequences of 7 amino acids bounded by cystines at the N- and C- terminals.  Further screening of the peptides was done by performing 6 successive  incubation-wash-elution cycles against HT-29. See <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1550331/pdf/neo0505_0437.pdf">Kelly &amp; Jones  (2003)</a>.  &lsquo;RPM&rsquo; - for an arginine-proline-methionine amino acid sequence immediately  before the C-terminal cystine - emerged as a consensus motif for late selection high-affinity peptides, thus giving the peptide&rsquo;s name. RPMrel&rsquo;s full amino  acid sequence is n-CPIEDRPMC-c. </p>
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<div align="left">
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<p>The binding properties of RPMrel  were identified during Kelly &amp; Jones&rsquo; study when it was fused to the  surface-exposed p3 minor coat protein of the bacteriophage M13KE. This module  will lead to its novel fusion to the cell wall binding domain of lytC, exposing  it to the extracellular environment. </p>
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<h1><p>Design</p></h1>
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<p><strong>lytC, and its cell wall binding domain.</strong></p>
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</div>
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<p>lytC, a cell surface hydrolase, is native to <em>B. subtilis</em> and binds non-covalently to its cell wall  interacting with it electrostatically. This property was previously determined  to make it superior for exposure of bound peptides. Furthermore, as compared to  other surface expression methods we investigated (including peptide expression  on an engineered S-layer), the lytC model is much better studied. </p>
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<p>According to a study by <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC262103/pdf/0628.pdf">Yamamoto et al (2003)</a> lytC is localized uniformly on <em>B. subtilis</em> cells grown past log phase, making it more ideal than the more specifically localized lytE and lytF for expression of RPMrel. It was further found that - compared to the others - lytC was particularly resistant to degradation by the cell surface protease WprA and extracellular protease Epr, both <em>B. subtilis </em>products. In the same  study, 3xFLAG (a <a href="http://www.sigmaaldrich.com/life-science/proteomics/recombinant-protein-expression/purification-detection/flag-system.html">standard peptide epitope</a> designed by Sigma-Aldrich Corp.) was successfully fused to the protein via a short linker and was successfully exposed to specific antibodies.</p>
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<p><b>Considering limitations.</b></p>
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<p>The full sequence of lytC is encoded in 1488bp, but its cell wall binding domain was isolated by Imperial College London&rsquo;s 2010 team as the region encoded by the first 954bp. This means the natural function of lytC - cell wall turnover and autolysis for cell growth and separation - is removed from the recombinant protein we will use. </p>
+
 
-
          </div>
+
<p>Design of a solution starts with considering existing limitations. Since this is iGEM, the clearest limitation was that the solution must be a biological one and thus must involve a living component. Only a certain set of living organisms lie within our reasonable capacity to engineer them, and of these we decided on <i>Bacillus subtilis</i> (see <a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Design_Chassis">Chassis</a> page).</p>
-
  <div id="paragraph3" class="bodyParagraphs">
+
 
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          <div align="left">
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<p>We then examined the treatment environment. Carcinomas of the colon protrude into the digestive tract and items within the tract can interact with them directly. We decided on the concept that our biological system would be ingested, then produce and release the drug in the vicinity of the tumor for direct action.</p>
-
              <h1>Parts Assembly</h1>
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          </div>
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<p>As a ligand in the Transforming Growth Factor β (TGF-β) signalling pathway, BMP2 had to be expressed in mature form to have any effect. It was thus decided that it would be synthesized within our chassis and then released into the external environment by secretion. Since the polypeptide sequence and conformation of BMP2 must be preserved, it was decided to bring the drug to the vicinity of the carcinoma by conferring the binding ability of RPMrel to the chassis as a whole.</p>
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          <p><strong>A GFP  reporter.</strong></p>
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<p>Preliminary work of generating a  reporter expression construct was done first. This reporter construct in  question was produced by PCR amplification of the sequence encoding for green  fluorescence protein (GFP) and double terminator from <a href="http://partsregistry.org/Part:BBa_E0840">BBa_E0840</a> with simultaneous attachment of the <em>B. subtilis </em>consensus  RBS embedded within the forward primer. </p>
+
<p>To do this, RPMrel had to be expressed on the cell surface in a functional form. We conducted research into several methods to do this on <i>B. subtilis</i> and concluded that cell wall expression of the peptide was ideal. Imperial College London’s 2010 team had performed that same task using the cell wall binding domain of the hydrolase lytC as their peptide anchor and a helical linker of their design. We decided to employ that same system for surface expression of RPMrel. <br>
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<p>The product of this reaction was  thus [consensus RBS + GFP + double terminator] and was inserted into pSB1C3 for  future use. When expressed at the same time as RPMrel fused to the lytC cell  wall binding domain the recombinant bacteria will resolve in green under UV  illumination. </p>
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<p><strong>Amplification  of BBa_K316037.</strong></p>
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<br><b>RPMrel, and colon tumor specific binding.</b></p>
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<p>Exploratory work began by taking  the construct submitted in <a href="http://partsregistry.org/Part:BBa_K316037">BBa_K316037</a> as the starting point. BBa_K316037, originally  submitted by Imperial College London&rsquo;s 2010 team, contains the following  regions in order: [Pveg promoter, spoVG RBS, cell wall binding domain of lytC, helical linker, elastase cleavage site, auto-inducing peptide and his tag]. The  regions we wanted include Pveg promoter through to helical linker only.</p>
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<p>RPMrel - a 9 amino acid peptide -  can be synthesized <em>de novo</em> by PCR when embedded within a primer. We  chose to codon-optimize the amino acid sequence for expression in <em>B.  subtilis</em> and embed the resultant 27 nucleotide sequence in the design of a  reverse primer. This reverse primer was then used in a PCR reaction that amplified  [Pveg + spoVG + lytC + linker] from BBa_K316037 and simultaneously attached the  sequence of RPMrel to the end of the linker.  </p>
+
<p>Using phage display to compile peptide libraries that confer specific binding to certain antigens is a now common way to come up with useful peptides. RPMrel, a 9 amino acid disulfide-constrained peptide, was screened out of the New England Biolabs <a href="http://www.neb.com/nebecomm/products/producte8100.asp">Ph.D.-C7C library</a> for positive binding  to poorly differentiated HT-29 cells, and negative binding to well  differentiated HCT 116 cells. All peptides in the Ph.D.-C7C library have random  sequences of 7 amino acids bounded by cystines at the N- and C- terminals.  Further screening of the peptides was done by performing 6 successive  incubation-wash-elution cycles against HT-29. See <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1550331/pdf/neo0505_0437.pdf">Kelly &amp; Jones  (2003)</a>.  &lsquo;RPM&rsquo; - for an arginine-proline-methionine amino acid sequence immediately  before the C-terminal cystine - emerged as a consensus motif for late selection of high-affinity peptides, thus giving the peptide&rsquo;s name. RPMrel&rsquo;s full amino  acid sequence is n-CPIEDRPMC-c.
-
<p>Design of that reverse primer is  detailed as follows. <br />
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  5' - [8bp cap] [7bp SpeI  restriction site] [6bp reverse-complementary double stop codon] [27bp  reverse-complementary sequence of codon optimized RPMrel] [15bp  reverse-complementary overlap with linker] - 3'</p>
+
<p>The binding properties of RPMrel  were identified during Kelly &amp; Jones&rsquo; study when it was fused to the  surface-exposed p3 minor coat protein of the bacteriophage M13KE. This module  will lead to its novel fusion to the cell wall binding domain of lytC, exposing  it to the extracellular environment. <br>
-
<p>The exact sequence is as follows.<br />
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  5&rsquo; -  GTTTCTTCACTAGTATTATTAACACATCGGGCGATCTTCGATCGGACAGGCCGCGGCTTTCGC - 3&rsquo; (63bp)</p>
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<br><strong>LytC, and its cell wall binding domain.</strong></p>
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<p><strong>Assembly  of PCR products.</strong></p>
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<p>The product of this PCR reaction  in linear form comprised [Pveg + spoVG + lytC + linker + RPMrel]. Standard  assembly methods were then used to join it in front of the aforementioned  [consensus RBS + GFP + double terminator] construct in pSB1C3. Following this  step, the module&rsquo;s code comprised [Pveg + spoVG + lytC + linker + RPMrel +  consensus RBS + GFP + double terminator]. The module&rsquo;s was submitted to the  Registry in this form (see <a href="http://partsregistry.org/Part:BBa_K733007">BBa_K733007</a>). </p>
+
<p>LytC, a cell surface hydrolase, is native to <em>B. subtilis</em> and binds non-covalently to its cell wall  interacting with it electrostatically. This property was previously determined  to make it superior for exposure of bound peptides. Furthermore, as compared to  other surface expression methods we investigated (including peptide expression  on an engineered S-layer), the LytC model was much better studied. </p>
-
<p>Further cloning work was done to  ligate the module&rsquo;s code into pDG1661, an integration vector for <em>B. subtilis</em> which was its final destination for characterization. For more details on  pDG1661, see <a href="http://www.bgsc.org/catpart4.pdf">this document</a> produced by the  Bacillus Genetic Stock Center (BGSC). </p>
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-
          </div>
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<p>According to the study conducted by <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC262103/pdf/0628.pdf">Yamamoto et al (2003)</a> LytC is localized uniformly on <em>B. subtilis</em> cells grown past log phase, making it more ideal than the more specifically localized LytE and LytF for expression of RPMrel. It was further found that - compared to the others - LytC was particularly resistant to degradation by the cell surface protease WprA and extracellular protease Epr, both products of <em>B. subtilis </em>. In this study, 3xFLAG (a <a href="http://www.sigmaaldrich.com/life-science/proteomics/recombinant-protein-expression/purification-detection/flag-system.html">standard peptide epitope</a> designed by Sigma-Aldrich Corp.) was successfully fused to the protein via a short linker and was successfully exposed to specific antibodies.</p>
-
  <div id="paragraph4" class="bodyParagraphs">
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          <div align="left">
+
<p>The full sequence of the <em>lytC</em> gene is 1488bp, but its cell wall binding domain was isolated by Imperial College London&rsquo;s 2010 team as the region encoded by the first 954bp. This means the natural function of LytC - cell wall turnover and autolysis for cell growth and separation - is removed from the recombinant protein we will use. </p>
-
              <h1>Testing (Characterization & Results)</h1>
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          </div>
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<p align="center"><img src="https://static.igem.org/mediawiki/2012/1/18/Cwbd.JPG" width="65%" /></p>
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          <p>BBa_K733007 is to result in the  production of two gene products: 1) the lytC + RPMrel fusion protein localized  to the cell wall, and 2) green fluorescence protein. <em>B. subtilis </em>cells  transformed with the construct are expected to exhibit both binding affinity  for HT-29 cells and green fluorescence under UV illumination. </p>
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<p>The module is to be tested by  washing fixed and stained HT-29 colorectal cancer cells with <em>B. subtilis  subtilis </em>168 transformed with the construct contained in <a href="http://partsregistry.org/Part:BBa_K733007">BBa_K733007</a> in mammalian cell  media. Following successive washes with phosphate buffered serum (PBS), the  fixed cells will be imaged by fluorescence microscopy at a magnification  resolving the HT-29 cells. </p>
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<p>The same process will be conducted  using <em>B. subtilis subtilis </em>168 cells transformed with only GFP as a  negative control. By comparing the relative localization of fluorescence  between the two images, the effect of the lytC + RPMrel fusion protein on the  recombinant bacteria&rsquo;s binding ability can be visualized. </p>
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<h1><p>References</p></h1>
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<p>Kelly, Kimberly A., and David A. Jones. "Isolation of a Colon Tumor Specific Binding Peptide Using Phage Display Selection." <i>Neoplasia</i> 5.5 (2003): 437-444. Print.</p>
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<p>Yamamoto, Hiroki, Shin-ichirou Kurosawa, and Junichi Sekiguchi. "Localization of the Vegetative Cell Wall Hydrolases LytC, LytE, and LytF on the Bacillus subtilis Cell Surface and Stability of These Enzymes to Cell Wall-Bound or Extracellular Proteases." <i>Journal of Bacteriology</i> 185.22 (2003): 6666-6677. Print.</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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Latest revision as of 00:36, 27 September 2012

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

Target Binding Module

<<< Back to Modules

Overview

Our decision to pursue colorectal carcinoma suppression arose from two key points obtained from preliminary research: 1) bone morphogenetic protein 2 (BMP2) suppresses the growth of colon cancer cell growth in vivo, and 2) the phage display peptide RPMrel confers specific and preferential binding to non-differentiated colon cancer cells.

With these two pieces of knowledge we have respectively: 1) our carcinoma suppression drug, and 2) a tool for specifically targeting cancerous cells. Thus the objective of this module was to identify and then construct a suitable mechanism making use of the RPMrel peptide to target the delivery of BMP2.

Design

Considering limitations.

Design of a solution starts with considering existing limitations. Since this is iGEM, the clearest limitation was that the solution must be a biological one and thus must involve a living component. Only a certain set of living organisms lie within our reasonable capacity to engineer them, and of these we decided on Bacillus subtilis (see Chassis page).

We then examined the treatment environment. Carcinomas of the colon protrude into the digestive tract and items within the tract can interact with them directly. We decided on the concept that our biological system would be ingested, then produce and release the drug in the vicinity of the tumor for direct action.

As a ligand in the Transforming Growth Factor β (TGF-β) signalling pathway, BMP2 had to be expressed in mature form to have any effect. It was thus decided that it would be synthesized within our chassis and then released into the external environment by secretion. Since the polypeptide sequence and conformation of BMP2 must be preserved, it was decided to bring the drug to the vicinity of the carcinoma by conferring the binding ability of RPMrel to the chassis as a whole.

To do this, RPMrel had to be expressed on the cell surface in a functional form. We conducted research into several methods to do this on B. subtilis and concluded that cell wall expression of the peptide was ideal. Imperial College London’s 2010 team had performed that same task using the cell wall binding domain of the hydrolase lytC as their peptide anchor and a helical linker of their design. We decided to employ that same system for surface expression of RPMrel.

RPMrel, and colon tumor specific binding.

Using phage display to compile peptide libraries that confer specific binding to certain antigens is a now common way to come up with useful peptides. RPMrel, a 9 amino acid disulfide-constrained peptide, was screened out of the New England Biolabs Ph.D.-C7C library for positive binding to poorly differentiated HT-29 cells, and negative binding to well differentiated HCT 116 cells. All peptides in the Ph.D.-C7C library have random sequences of 7 amino acids bounded by cystines at the N- and C- terminals. Further screening of the peptides was done by performing 6 successive incubation-wash-elution cycles against HT-29. See Kelly & Jones (2003). ‘RPM’ - for an arginine-proline-methionine amino acid sequence immediately before the C-terminal cystine - emerged as a consensus motif for late selection of high-affinity peptides, thus giving the peptide’s name. RPMrel’s full amino acid sequence is n-CPIEDRPMC-c.

The binding properties of RPMrel were identified during Kelly & Jones’ study when it was fused to the surface-exposed p3 minor coat protein of the bacteriophage M13KE. This module will lead to its novel fusion to the cell wall binding domain of lytC, exposing it to the extracellular environment.

LytC, and its cell wall binding domain.

LytC, a cell surface hydrolase, is native to B. subtilis and binds non-covalently to its cell wall interacting with it electrostatically. This property was previously determined to make it superior for exposure of bound peptides. Furthermore, as compared to other surface expression methods we investigated (including peptide expression on an engineered S-layer), the LytC model was much better studied.

According to the study conducted by Yamamoto et al (2003) LytC is localized uniformly on B. subtilis cells grown past log phase, making it more ideal than the more specifically localized LytE and LytF for expression of RPMrel. It was further found that - compared to the others - LytC was particularly resistant to degradation by the cell surface protease WprA and extracellular protease Epr, both products of B. subtilis . In this study, 3xFLAG (a standard peptide epitope designed by Sigma-Aldrich Corp.) was successfully fused to the protein via a short linker and was successfully exposed to specific antibodies.

The full sequence of the lytC gene is 1488bp, but its cell wall binding domain was isolated by Imperial College London’s 2010 team as the region encoded by the first 954bp. This means the natural function of LytC - cell wall turnover and autolysis for cell growth and separation - is removed from the recombinant protein we will use.

References

Kelly, Kimberly A., and David A. Jones. "Isolation of a Colon Tumor Specific Binding Peptide Using Phage Display Selection." Neoplasia 5.5 (2003): 437-444. Print.

Yamamoto, Hiroki, Shin-ichirou Kurosawa, and Junichi Sekiguchi. "Localization of the Vegetative Cell Wall Hydrolases LytC, LytE, and LytF on the Bacillus subtilis Cell Surface and Stability of These Enzymes to Cell Wall-Bound or Extracellular Proteases." Journal of Bacteriology 185.22 (2003): 6666-6677. Print.

Anti-tumor Molecule Secretion Module

Regulation and Control Module

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