Difference between revisions of "Team:Tokyo Tech/The experiments of creating E. coli mutants which secrete protein extracellularly"

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<h1 align="center">The experiments of creating E. coli mutants which secrete protein extracellularly</h1>
 
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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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    <h2><span>Contents</span></h2>
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<h3 class="link"><a href="#">1. unnun</a></h3><!--右にある#の後に入れたIDを、下の"ID"の中に入れてな-->
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    <h3 class="link"><a href="#introduction">1. Introduction</a></h3>
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    <h3 class="link"><a href="#summary">2. Summary of the experiment</a></h3>
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    <h3 class="link"><a href="#results">3. Results</a></h3>
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    <h3 class="link"><a href="#methods">4. Materials and methods</a></h3>
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    <h3 class="link"><a href="#construction"><font size="2.7">&nbsp;&nbsp;&nbsp;4-1. Construction</font></a></h3>
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    <h3 class="link"><a href="#protocol"><font size="2.7">&nbsp;&nbsp;&nbsp;4-2. Assay protocol</font></a></h3>
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    <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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    <div align="center"><img src="https://static.igem.org/mediawiki/2016/d/d2/Fig.5-2-3_3.jpg" height ="200"><br></div>
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<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 5-2-3 </span>
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    <h2><span>4. Material and methods</span></h2>
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    <p class="normal_text">
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<h3 algin>4-1. Construction</h3></p>
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<h4 algin>-Strain</h4>
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  <p class="normal_text">All the plasmids were prepared in MG1655 strain.</p>
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  <p class="normal_text">DH5alpha</p>
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<h4 algin>-Plasmid</p></h4>
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<p class="normal_text">A. Pcon-rfp (pSB6A1)</p>
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<h4 algin>-Medium</h4>
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  <p class="normal_text">LB medium A:</p>
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  <p class="normal_text">LB medium containing ampicillin (50 microg/ mL) </p>
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  <p class="normal_text"><h3 align>4-2. Assay protocol</h3></p>
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  <p class="normal_text">The following experiments is performed at 37℃ unless otherwise stated.</p>
 +
 
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  <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>
 +
 
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  <p class="normal_text">3) Streak the fresh cultures on an agar plate containing ampicillin (50 microg/ mL).</p>
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  <p class="normal_text">4) Expose the plate at 9 or 11 *100 microJ/ cm2 and incubate it overnight.</p>
 +
 
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  <p class="normal_text">5) Make a replica of the colonies on the plate with velvet.</p>
 +
 
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  <p class="normal_text">6) Pore the soft agar containing ampicillin (50 microg/ mL).</p>
 +
 
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  <p class="normal_text">7) Streak DH5alpha without ampicillin resistance on the soft agar and incubate overnight.</p>
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  <p class="normal_text">8) Observe whether the colonies of DH5alpha are on the soft agar.
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    <h2><span>5. Reference</span></h2>
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<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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Latest revision as of 21:08, 19 October 2016

E. coli mutation secreting proteins extracellularly

1. Introduction

E. coli has the two membrane: the outer membrane and the inner membrane. Then, E. coli rarely secrets proteins to outside the cell.

2. Summary of the experiment

To put our future work into practice, it is essential to obtain E. coli having the ability of secreting any favorite protein. To obtain such E. coli, we expose UV to MG1655 containing beta-lacetamase, 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.

3. Results

The pictures of the exposed UV colonies below (Fig.5-2-3).


Fig. 5-2-3


4. Material and methods

4-1. Construction

-Strain

All the plasmids were prepared in MG1655 strain.

DH5alpha

-Plasmid

A. Pcon-rfp (pSB6A1)

-Medium

LB medium A:

LB medium containing ampicillin (50 microg/ mL)

4-2. Assay protocol

The following experiments is performed at 37℃ unless otherwise stated.

1) Incubate transformed MG1655 containing plasmid-A with vigorous shaking overnight.

2) Dilute the overnight cultures to 1/100,000 in fresh LB medium A.

3) Streak the fresh cultures on an agar plate containing ampicillin (50 microg/ mL).

4) Expose the plate at 9 or 11 *100 microJ/ cm2 and incubate it overnight.

5) Make a replica of the colonies on the plate with velvet.

6) Pore the soft agar containing ampicillin (50 microg/ mL).

7) Streak DH5alpha without ampicillin resistance on the soft agar and incubate overnight.

8) Observe whether the colonies of DH5alpha are on the soft agar.

5. Reference

(1) F.J.M. Mergulha ̃o et al (2005) Recombinant protein secretion in Escherichia coli. Biotechnology Advances 23 (2005) 177–202