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

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     <h2><span>4. Material and methods</span></h2>
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     <h2><span>4. Material & methods</span></h2>
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4-1. Construction
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<h3 algin>4-1. Construction</h3></p>
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  -Strain
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  <h4 algin>-Strain</h4>
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   All the plasmids were prepared in MG1655 strain.
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   <p class="normal_text">All the plasmids were prepared in MG1655 strain.</p>
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   DH5a
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  A. Pcon-rfp (pSB6A1)
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-Medium
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LB medium A:
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   LB medium containing ampicillin (50 microg/ mL)  
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   <p class="normal_text">LB medium containing ampicillin (50 microg/ mL) </p>
  
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4-2. Assay Protocol
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  <p class="normal_text"><h3 align>4-2. Assay protocol</h3></p>
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The following experiments is performed at 37℃ unless otherwise stated.
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  <p class="normal_text">The following experiments is performed at 37℃ unless otherwise stated.</p>
  
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1) Incubate transformed MG1655 containing plasmid-A with vigorous shaking overnight.
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  <p class="normal_text">1) Incubate transformed MG1655 containing plasmid-A with vigorous shaking overnight.</p>
  
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2) Dilute the overnight cultures to 1/100,000 in fresh LB medium A.
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  <p class="normal_text">2) Dilute the overnight cultures to 1/100,000 in fresh LB medium A.</p>
  
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3) Streak the fresh cultures on an agar plate containing ampicillin (50 microg/ mL).
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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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4) Expose the plate at 9 or 11 *100 microJ/ CM2 and incubate it overnight.
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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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5) Make a replica of the colonies on the plate with velvet.
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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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6) Pore the soft agar containing ampicillin (50 microg/ mL).
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  <p class="normal_text">6) Pore the soft agar containing ampicillin (50 microg/ mL).</p>
  
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7) Streak DH5a without ampicillin resistance on the soft agar and incubate overnight.
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  <p class="normal_text">7) Streak DH5a 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 DH5a 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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8) Observe whether the colonies of DH5a are on the soft agar.
 
  
  

Revision as of 05:20, 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).

4. Material and methods

4-1. Construction

-Strain

All the plasmids were prepared in MG1655 strain.

DH5a

-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 DH5a without ampicillin resistance on the soft agar and incubate overnight.

8) Observe whether the colonies of DH5a 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