Difference between revisions of "Team:Ionis Paris/Notebook/18 10 16bis"

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                                     <h3><font color =”94FAF1”> Interpretation</font></h3>   
 
                                     <h3><font color =”94FAF1”> Interpretation</font></h3>   
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<p>TWe optained red colonies which prove that the mRFP is synthesized by the cells and in the same way the XylR protein.
 
<p>TWe optained red colonies which prove that the mRFP is synthesized by the cells and in the same way the XylR protein.
 
We have shown that our E.Coli DH5⍺ transformed with the biobrick BBa_K2023013 synthesized XylR in a constitutive way.</p>  
 
We have shown that our E.Coli DH5⍺ transformed with the biobrick BBa_K2023013 synthesized XylR in a constitutive way.</p>  
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                                        <h2 class="blog_topHd"> <font color =”#279AD3”>Competent cells: E.Coli DH5?</font></h2>
 
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                                    <h3><font color =”94FAF1”> Objectives </font></h3>                   
 
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            <p>The objective is to make a stock of E.Coli DH5? competent cells for subsequent transformations.</p>
 
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                                  <h3><font color =”94FAF1”> Materials </font></h3>
 
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<ul><li>O/N DH5? pre-culture (made the 22/07): O/N inoculation of 100 µL DH5? into 100 mL LB.</li>
 
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<li>0.1M CaCl2: prepared the 23/07</li>
 
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<li>0.1M CaCl2/15% Glycerol: prepared on the 23/07</li></ul>
 
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                                <h3><font color =”94FAF1”> Protocol </font></h3>   
 
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  <h5><font color =”#3CB5E1”> Competence: </font></h5>
 
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    <li>Inoculation of 3 mL O/N culture in 100 mL LB in 500 mL erlenmeyer.</li>
 
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    <li>Incubation at 250 rpm at 37°C until the DO 0,6 —> DO = 0.591</li>
 
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    <li>Cells were transferred to 3 sterile ice-cold 50 mL Falcon tubes. 20 mL in each falcon tube.</li>
 
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    <li>Incubate on ice for 30 min. Do not allow cells to warm up over 4°C.</li>
 
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    <li>Spin cells at 4000 rpm for 10 min at 4°C.</li>
 
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    <li>Discard supernatant and try to drain all remaining media.</li>
 
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    <li>Gently resuspend on 10 mL cold 0.1M CaCl2</li>
 
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    <li>Incubate on ice for 20 min</li>
 
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    <li>Centrifuge 10 min at 4,000 rpm at 4°C</li>
 
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    <li>Discard supernatant and gently resuspend on 5 mL cold 0.1M CaCl2/25% Glycerol</li>
 
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    <li>Transfer in 1.5 mL eppendorf (100 µL/tube)</li>
 
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    <li>Store at -80°C</li>
 
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</ol>
 
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<p>NB: The competency of the prepared cells will be tested on the 20/09.</p>
 
  
  

Latest revision as of 03:35, 20 October 2016

XylR Synthesis Test on BBX1

Objectives

The overall purpose is to check if our BBX1 biobrick works in E.Coli DH5⍺. This biobrick is composed with XylR coding device with mRFP (Pr-RBS-XylR-RBS-mRFP-Terminator) and has been designed for the monitoring of XylR and to show that this protein is synthesized in the cells when the colonies becomes red thanks to the mRFP protein.

Materials

  • BBX1: "XylR coding device with mRFP”, BBa_K2023013 (Pr-RBS-XylR-RBS-mRFP-Terminator).

  • E.Coli DH5⍺ from the -80°C transformed with X1.

  • SOC medium at room temperature.

Protocol

  1. Let the cells on ice 10 minutes for the defrosting.

  2. Add SOC medium in order to have a dilution of 1/2, 1/10 and 1/100.

  3. Plate the different concentrations (Without dilution and the 3 dilutions) on petri dish with LB Agare + Chloramphenicol at 25 µg/mL final.

Results

Expected results:

We expected red colonies due to the synthesis of the red protein mRFP. The promoter of this protein is Pr, a strong constitutive promoter.

Obtained results:

We obtained expected results.

Interpretation

TWe optained red colonies which prove that the mRFP is synthesized by the cells and in the same way the XylR protein. We have shown that our E.Coli DH5⍺ transformed with the biobrick BBa_K2023013 synthesized XylR in a constitutive way.

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