View source for Team:Tokyo Tech/The experiments of creating E. coli mutants which secrete protein extracellularly

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   <h1 align="center"><i>E. coli</i> mutation secreting proteins extracellularly</h1>
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    <h2><span>Contents</span></h2>
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    <h3 class="link"><a href="#introduction">1. Introduction</a></h3>
    <h3 class="link"><a href="#summary">2. Summary of the experiment</a></h3>
    <h3 class="link"><a href="#results">3. Results</a></h3>
    <h3 class="link"><a href="#methods">4. Materials and methods</a></h3>
    <h3 class="link"><a href="#construction"><font size="2.7">&nbsp;&nbsp;&nbsp;4-1. Construction</font></a></h3>
    <h3 class="link"><a href="#protocol"><font size="2.7">&nbsp;&nbsp;&nbsp;4-2. Assay protocol</font></a></h3>
    <h3 class="link"><a href="#reference">5. Reference</a></h3>
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    <h2><span>1. Introduction</span></h2>
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    <p class="normal_text">  <i>E. coli</i> has the two membrane: the outer membrane and the inner membrane. Then, <i>E. coli</i> rarely secrets proteins to outside the cell. 

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    <h2><span>2. Summary of the experiment</span></h2>
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    <p class="normal_text"> To put our future work into practice, it is essential to obtain <i>E. coli</i> having the ability of secreting any favorite protein. To obtain such <i>E. coli</i>, we expose UV to MG1655 containing <i>beta-lacetamase</i>, which has signal peptide and be secreted to periplasm. We are working on trying to obtain the mutant having ability of secreting protein to outside the cell.

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    <h2><span>3. Results</span></h2>
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    <p class="normal_text"> The pictures of the exposed UV colonies below (Fig.5-2-3). <br>


                                       
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    <h2><span>4. Material and methods</span></h2>
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<h3 algin>4-1. Construction</h3></p>
 <h4 algin>-Strain</h4>
   <p class="normal_text">All the plasmids were prepared in MG1655 strain.</p>
   <p class="normal_text">DH5a</p>

 <h4 algin>-Plasmid</p></h4>
 <p class="normal_text">A. Pcon-rfp (pSB6A1)</p>
 
<h4 algin>-Medium</h4>
   <p class="normal_text">LB medium A:</p>
  <p class="normal_text">LB medium containing ampicillin (50 microg/ mL) </p>

  <p class="normal_text"><h3 align>4-2. Assay protocol</h3></p>
  <p class="normal_text">The following experiments is performed at 37℃ unless otherwise stated.</p>

  <p class="normal_text">1) Incubate transformed MG1655 containing plasmid-A with vigorous shaking overnight.</p>

  <p class="normal_text">2) Dilute the overnight cultures to 1/100,000 in fresh LB medium A.</p>

  <p class="normal_text">3) Streak the fresh cultures on an agar plate containing ampicillin (50 microg/ mL).</p>

  <p class="normal_text">4) Expose the plate at 9 or 11 *100 microJ/ CM2 and incubate it overnight.</p>

  <p class="normal_text">5) Make a replica of the colonies on the plate with velvet.</p>

  <p class="normal_text">6) Pore the soft agar containing ampicillin (50 microg/ mL).</p>

  <p class="normal_text">7) Streak DH5a without ampicillin resistance on the soft agar and incubate overnight.</p>

  <p class="normal_text">8) Observe whether the colonies of DH5a are on the soft agar.


                                       
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    <h2><span>5. Reference</span></h2>
<p class="normal_text">(1) F.J.M. Mergulha ̃o et al (2005) Recombinant protein secretion in Escherichia coli. Biotechnology Advances 23 (2005) 177–202 </p>

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