Difference between revisions of "Team:KU Leuven/InterLabStudy/Protocol"

 
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<div class="summaryheader">
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  <div class="summaryimg">
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            <div class="summaryimg">
  <img src="https://static.igem.org/mediawiki/2015/e/eb/KU_Leuven_fossilBackground.png" width="100%">
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                <img src="https://static.igem.org/mediawiki/2015/c/c0/KU_Leuven_Banner_Rood5.jpg"
  <div class="head">
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    <h2> Protocol </h2>
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                <div class="head">
  </div>
+
                    <h2>
</div>
+
                        Protocols
</div>
+
                    </h2>
 +
                </div>
 +
            </div>
 +
        </div>
  
<!----------------------------Scientific !---------------------------->
+
        <!----------------------------Scientific !---------------------------->
  
<div class="summarytext1">
+
        <div class="summarytext1">
<div class="part">
+
            <div class="part">
  
<h2> Introduction </h2>
+
                <h2>
<p>
+
                    Introduction
We began our experiments by constructing devices that contained constitutive promoters with low (J23117), medium (J23106) and higher (J23101) levels of GFP expression. Each device contains the biobrick I13504, necessary for GFP expression. We transformed the above mentioned biobrick and the promoters in E. cloni competent cells.  
+
                </h2>
The cells were grown on a LB (from Sigma) 1.5% agar (from VWR Chemicals) plates with chloramphenicol (from Acros Organics) as a selection marker. As a positive control, we transformed the cells with pUC19 plasmid and plated them on LB plates containing ampicillin. We also plated cells without any plasmid as a negative control on LB plates containing chloramphenicol. We performed transformation of the biobricks twice by using chemically competent cells. The first time, we did not obtain any colonies of the four biobricks. The second time we got very few colonies. Nevertheless, the positive controls were correct every time, and we did double check the efficiency of the cells that proved to be very high. We concluded that our constructs were not easy to transform the bacteria. Therefore, to have more effective transformation, we switched to electroporation. This technique gave a higher efficiency and enough colonies for our experiments.
+
                <p>
</p>
+
                    Experiments started with the construction of devices that contained constitutive
<br>
+
                    promoters with low (J23117), medium (J23106) and higher (J23101) strength.
<p>
+
                    Each promoter was coupled to BioBrick I13504, containing a RBS, GFP protein and a double terminator.
Thereafter we proceeded using the Biobrick Assembly Method to assemble the DNA. Subsequently we performed transformation using electrocompetent E.cloni cells, plated them in LB agar plates with antibiotic selection markers, and the plates were illuminated with blue/UV-light to check for the presence of GFP, and thus the functioning device.  
+
                    The above mentioned BioBrick and the promoters were transformed in <i>E. cloni</i> competent cells. The cells were grown on LB (Sigma-Aldrich) 1.5% agar (VWR Chemicals) plates with chloramphenicol (from Acros Organics) as a selection
</p>
+
                    marker. As a positive control, cells were also transformed with the pUC19 plasmid and
<br>
+
                    plated on LB plates containing ampicillin. <i>E. cloni</i> without any
<p>
+
                    plasmid was also plated as a negative control on LB plates containing chloramphenicol.  
For the fluorescent measurements we inoculated liquid cultures(3 mL-LB+Antibiotic) in polypropylene round-bottom tubes and incubated them for 16 to 18 hours in a shaking incubator (200 rpm) at 37 degrees. We recorded the fluorescent data from cells grown to an OD of ~0.5 (if the OD is higher bring it in the range 0.48-0.52) at 300 nm. Finally, the fluorescence data were collected from the overnight cultures of the constructed devices with an excitation and emission wavelengths of 483 nm and 525 nm respectively, in a 96-well plate by an Tecan Safire2 monochromator MTP Reader. Also, the absorbance measurements at 600 nm were repeated in the plate reader. This is important because the absorbance depends on the path length.
+
                    Transformation of the BioBricks was performed twice by using chemically competent
</p>
+
                    cells. The first time, no colonies from any of the four BioBricks were obtained. The
 +
                    second time, only a few colonies grew. Nevertheless, the positive controls were
 +
                    correct every time and the transformation efficiency of our <i>E. cloni</i> was previously proven to be very high. Therefore, we switched to electroporation. This technique showed a higher efficiency and enough
 +
                    colonies grew to perform the measurements.
 +
                    <br></br>
 +
 
 +
                    Thereafter, the BioBrick Assembly Method was used to combine the promoters with GFP.
 +
                    Subsequently, electrocompetent <i>E. cloni</i> cells were transformed,
 +
                    plated on LB agar plates with antibiotic selection markers, illuminated with blue/UV-light to check for the presence of GFP, and thus
 +
                    a functional device.
 +
 
 +
                </br>
 +
 
 +
                For the fluorescent measurements, liquid cultures (3 mL-LB + Antibiotic) were inoculated in polypropylene round-bottom tubes and incubated for 16
 +
                to 18 hours in a shaking incubator (200 rpm) at 37 °C. The
 +
                fluorescence data from cells grown to an OD of ~0.5 (if the OD was higher, it was brought
 +
                in the range 0.48-0.52) were measured at 300 nm. Finally, the fluorescence data were collected
 +
                from the overnight cultures of the constructed devices with excitation and
 +
                emission wavelengths of 483 nm and 525 nm respectively in a 96-well plate by a
 +
                Tecan Safire2 monochromator MTP Reader. Besides, the absorbance measurements at 600
 +
                nm were repeated in the plate reader to normalize for cell density.  
 +
            </br>
 +
        </p>
 +
    </div>
 +
    <div class="part">
 +
 
 +
        <h2>
 +
            Methodology</h2>
 +
    </div>
 +
<div class="center">
 +
    <div class="togglebar">
 +
        <div class="toggleone">
 +
            <h2>Preparing electrocompetent cells</h2>
 +
        </div>
 +
      <div id="toggleone">
 +
            <dl>
 +
              <dd>- Make a liquid culture of a single colony in 1-3 mL salt free LB</dd>
 +
        <dd>- Grow 300-400 mL cells (without salt) at 37 °C until the O.D. reaches 0.6</dd>
 +
  <dd>- Cool down on ice and perform all the steps at 4 °C</dd>
 +
<dd>- Spin the cells down in falcon tubes (3500 g, 20 min, 4 °C)</dd>
 +
<dd>- Resuspend the cells in 10% glycerol, spin the cells down (5000 g, 10 min, 4 °C). Repeat this step 3 times</dd>
 +
<dd>- Resuspend the cells in 10% glycerol to obtain a dense pulp (usually not more
 +
than 1.5 mL)</dd>
 +
<dd>- Take 50 µL sample and do the electroporation test (without DNA). Pulses should be
 +
between 4 and 6 msec. If shorter, wash the cells once again with 30 mL
 +
glycerol</dd>
 +
<dd>- Aliquot the cells (50 µL), quick-freeze in liquid nitrogen and store at -80 °C</dd>
 +
</dl>
 
</div>
 
</div>
 
</div>
 
</div>
 +
</br>
  
<div class="summarytext2">
+
<div class="togglebar">
<div class="part">
+
<div class="toggletwo">
<div>
+
<h2>Electroporation</h2>
<h3> Methodology</h3>
+
 
</div>
 
</div>
 +
<div id="toggletwo" >
 +
<dl>
 +
<dd>- Add 1 µl DNA to 50 µl electrocompetent cells in an ice-cold cuvette (1 mm)</dd>
 +
<dd>- Electroporate (Eppendorf, 1700 V, 4 msec)</dd>
 +
<dd>- Add 950 µl of SOC solution</dd>
 +
<dd>- Incubate for one hour at 37 °C</dd>
 +
<dd>- Plate out on pre-warmed plates containing the correct selective medium, in this case chlormaphenicol for J23101, J23106 and J23117 and ampicillin for I13504 (37 °C)</dd>
 +
</dl>
 
</div>
 
</div>
 
<div class="example">
 
<div class="one">
 
<h2>Preparing electrocompetent cells</h2>
 
 
</div>
 
</div>
 +
</br>
 +
<div class="togglebar">
 +
<div class="togglethree">
 +
<h2>BioBrick Assembly Method</h2>
 
</div>
 
</div>
 
+
<div id="togglethree" >
<div id="one">
+
<dl>
<div class="methodology">
+
<dd>- Digest I13504 (GFP) with XbaI and PstI in Tango buffer</dd>
1. Make a liquid culture of a single colony in 1-3 mL salt free LB</br>
+
<dd>- Digest the promoters J23101, J23106 and J23117 with PstI in buffer O</dd>
2. Grow 300-400 mL cells (without salt) in 37°C until the O.D.reaches 0.6</br>
+
<dd>- Load the digested I13504 on a 1.5% agarose gel and visualize it under UV light.</dd>
3. Cool down on ice and from now on perform all the steps at 4 °C</br>
+
<dd>- Thereafter, perform a gel purification of I13504 (GeneJET Gel Extraction Kit -
4. Spin the cells down in falcon tubes (3500 g, 20 min, 4°C)</br>
+
- ThermoFisher Scientific)</dd>
5. Resuspend the cells in 10 % glycerol, spin the cells down (5000 g, 10 min, 4 °C). Repeat this step 3 times</br>
+
<dd>- PCR purify the promoters J23101, J23106 and J23117</dd>
6. Resuspend the cells in 10 % glycerol to obtain a dense pulp (usually not more than 1.5 mL)</br>
+
<dd>- Digest the promoters J23101, J23106 and J23117 with FD SpeI in 10x Fast Digest
7. Take 50 µL sample and do the electroporation test (without DNA). You should have a pulse of 4-6 msec. If it is shorter, wash the cells once again with 30 mL glycerol</br>
+
Buffer</dd>
8. Aliquot the cells (50 µL) and quick-freeze in liquid nitrogen and store at -80 °C</br>
+
<dd>- Ligate every promoter with I13504 using T4 DNA ligase</dd>
 +
</dl>
 
</div>
 
</div>
 
</div>
 
</div>
 +
</br>
  
<div class="example">
+
<div class="togglebar">
<div class="two">
+
<div class="togglefour">
<h2>This is example two</h2>
+
<h2>Restriction Mapping</h2>
 
</div>
 
</div>
<div id="two">
+
<div id="togglefour">
pompompom<br/>
+
<dl>
pompompom<br/>
+
<dd>- Digest with NcoI (cuts 1x in pSB1C3) and XhoI (cuts 1x in GFP) in Tango buffer</dd>
pompompom<br/>
+
<dd>- Mix gently and spin down</dd>
 +
<dd>- Incubate for 2 hours at 37 °C in a heating block</dd>
 +
<dd>- Separate the fragments using gel electrophoresis on a 1.5% agarose gel</dd>
 +
</dl>
 
</div>
 
</div>
 
</div>
 
</div>
 +
</br>
  
<div class="example">
 
<div class="three">
 
<h2>This is example thre</h2>
 
 
</div>
 
</div>
<div id="three">
+
<div class="part">
tiralalalala <br/>
+
                <h2>
tiralalala <br/>
+
                    Worksheet
tiralalala<br/>
+
                </h2>
 +
                <p>
 +
                  Our wetlab team worked well together to fulfill this challenge. Vincent Van Deuren and Laurens Vandebroek performed the BioBrick assembly and the transformation experiments. The measurements were recorded by Laetitia Van Wonterghem, Ovia Margaret Thirukkumaran and Laurens Vandebroek. Laura Van Hese, Astrid Deryckere, Ines Cottignie and Vincent Van Deuren carried out the restriction digestion to check for the inserts. Finally, the results were processed by Ovia Margaret Thirukkumaran and Laurens Vandebroek and our wiki-page was filled with provided data by Vincent Van Deuren and Laetitia Van Wonterghem. Our supervisor Katarzyna Malczewska coordinated the overall works and the rest of the team members served with a helping hand whenever needed.</br>
 +
 
 +
<br> To grow our cells, we made use of a New Brunswick Innova® 43/43R Shaker purchased from Eppendorf. This incubator has a throw of 2.54 cm. Our devices were measured by a Tecan Safire2 monochromator MTP Reader. This machine was last calibrated on the 31<sup>th</sup> of March in 2015 by Tecan and our measurements took place on the 25<sup>th</sup> of August in 2015. The cells were excited at 483 nm and the emission was recorded at 525 nm. To capture the light emission, a Quad4 Monochromator was used. The absorbance was measured at 600 nm with a sampling frequency of 0.11 seconds/ sample while the sampling frequency of the fluorescence was 0.15 seconds/sample.
 +
 
 +
            </br>
 +
        </p>
 +
    </div>
 
</div>
 
</div>
 
</div>
 
</div>
 +
<!--Foot don't touch!-->
  
 +
<div class="whiterow"></div>
  
<div class="example">
+
<div class="subsections">
<div class="four">
+
<div class="subsectionwrapper">
<h2>This is exaple one</h2>
+
<div class="subimgrow">
 +
<div class="whitespaceside"></div>
 +
<div class="subimg">
 +
<a href="https://2015.igem.org/Team:KU_Leuven/InterLabStudy/Results">
 +
<img src="https://static.igem.org/mediawiki/2015/e/e0/KU_Leuven_Wiki_Button_-_Results2.png" width="100%">
 +
</a>
 
</div>
 
</div>
<div id="four">
+
 
tiralalalala <br/>
+
<div class="whitespace">
tiralalala <br/>
+
tiralalala<br/>
+
 
</div>
 
</div>
 +
 +
<div class="subimg">
 +
<a href="https://2015.igem.org/Team:KU_Leuven/InterLabStudy">
 +
<img src="https://static.igem.org/mediawiki/2015/c/cb/KUL_Wiki_Button_-_Back.png" width="100%">
 +
</a>
 +
</div>
 +
<div class="whitespaceside"></div>
 
</div>
 
</div>
  
<div class="example">
+
<div class="subtextrow">
<div class="five">
+
<div class="whitespaceside"></div>
<h2>This is exaple one</h2>
+
<div class="subtext">
 +
<a href="https://2015.igem.org/Team:KU_Leuven/InterLabStudy/Results">
 +
<h2>Results</h2>
 +
<p>Click here to discover our results.</p>
 +
</a>
 
</div>
 
</div>
<div id="five">
+
 
tiralalalala <br/>
+
<div class="whitespace">
tiralalala <br/>
+
tiralalala<br/>
+
 
</div>
 
</div>
 +
 +
<div class="subtext">
 +
<a href="https://2015.igem.org/Team:KU_Leuven/InterLabStudy">
 +
<h2>Back</h2>
 +
<p>Go back to the Interlab page.</p>
 +
</a>
 +
</div>
 +
<div class="whitespaceside"></div>
 
</div>
 
</div>
  
 +
<div class="subimgreadmore">
 +
<div class="whitespaceside"></div>
 +
<div class="subimgrm">
 +
<a href="https://2015.igem.org/Team:KU_Leuven/InterLabStudy/Results">
 +
<div id="more">
 +
<img src="https://static.igem.org/mediawiki/2015/7/73/KUL_Wiki_Button_-_Read_more.png" height="40%" width="85%" alt="Read more">
 
</div>
 
</div>
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</a>
 
</div>
 
</div>
  
 +
<div class="whitespace"></div>
  
 +
<div class="subimgrm">
 +
<a href="https://2015.igem.org/Team:KU_Leuven/InterLabStudy">
 +
<div id="back">
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<img src="https://static.igem.org/mediawiki/2015/7/73/KUL_Wiki_Button_-_Read_more.png" height="40%" width="85%" alt="Read more">
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</div>
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</a>
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</div>
 +
<div class="whitespaceside"></div>
 +
</div>
  
 
+
<div class="subimgrowm">
<!--Foot don't touch!-->
+
<div class="subimgm">
 
+
<a href="https://2015.igem.org/Team:KU_Leuven/InterLabStudy/Results">
 
+
<b>Results</b>
<div class="subsections">
+
<img src="https://static.igem.org/mediawiki/2015/e/e0/KU_Leuven_Wiki_Button_-_Results2.png" width="100%">
<div class="subsectionwrapper">
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</a>
<div class="subimgrow">
+
</div>
 
+
  
 
<div class="whitespace">
 
<div class="whitespace">
 
</div>
 
</div>
  
<div class="subimg">
+
<div class="subtextm">
<a href="https://2015.igem.org/Team:KU_Leuven/Modeling">
+
<a href="https://2015.igem.org/Team:KU_Leuven/InterLabStudy/Results">
  <!---<img src="https://static.igem.org/mediawiki/2015/2/2b/KU_Leuven_Monocotyl.jpg" width="100%" height="100%" >-->
+
  <p>Click here to discover our results.<br/></p>
 
</a>
 
</a>
 
</div>
 
</div>
 
</div>
 
</div>
  
 +
<div class="subimgrowm">
 +
<div class="whiterow">
 +
</div>
 +
</div>
  
<div class="whitespace">
+
<div class="subimgrowm">
 +
<div class="subimgm">
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<a href="https://2015.igem.org/Team:KU_Leuven/InterLabStudy">
 +
<b>Back</b>
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<img src="https://static.igem.org/mediawiki/2015/c/cb/KUL_Wiki_Button_-_Back.png" width="100%" >
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</a>
 
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<div class="subtext">
+
<div class="whitespace"></div>
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<a href="https://2015.igem.org/Team:KU_Leuven/InterLabStudy">
 
<a href="https://2015.igem.org/Team:KU_Leuven/InterLabStudy">
  <h2>Back</h2>
+
  <p>Go back to the Interlab page.<br/></p>
 
</a>
 
</a>
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</div>
 
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<div style="height:40px;"></div>
 
  
 
<div id="summarywrapper">
 
<div id="summarywrapper">
 
<div class="summary">
 
<div class="summary">
<h3> Contact </h3>
+
<h3>
 +
Contact
 +
</h3>
 
<p style="font-size:1.3em; text-align: center">
 
<p style="font-size:1.3em; text-align: center">
 
Address: Celestijnenlaan 200G room 00.08 - 3001 Heverlee<br>
 
Address: Celestijnenlaan 200G room 00.08 - 3001 Heverlee<br>
Telephone : +32(0)16 32 73 19<br>
+
Telephone: +32(0)16 32 73 19<br>
Mail: igem@chem.kuleuven.be<br>
+
Email: igem@chem.kuleuven.be<br>
 
</p>
 
</p>
 
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<div id="footer">
 
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Latest revision as of 11:24, 10 November 2015

Protocols

Introduction

Experiments started with the construction of devices that contained constitutive promoters with low (J23117), medium (J23106) and higher (J23101) strength. Each promoter was coupled to BioBrick I13504, containing a RBS, GFP protein and a double terminator. The above mentioned BioBrick and the promoters were transformed in E. cloni competent cells. The cells were grown on LB (Sigma-Aldrich) 1.5% agar (VWR Chemicals) plates with chloramphenicol (from Acros Organics) as a selection marker. As a positive control, cells were also transformed with the pUC19 plasmid and plated on LB plates containing ampicillin. E. cloni without any plasmid was also plated as a negative control on LB plates containing chloramphenicol. Transformation of the BioBricks was performed twice by using chemically competent cells. The first time, no colonies from any of the four BioBricks were obtained. The second time, only a few colonies grew. Nevertheless, the positive controls were correct every time and the transformation efficiency of our E. cloni was previously proven to be very high. Therefore, we switched to electroporation. This technique showed a higher efficiency and enough colonies grew to perform the measurements.

Thereafter, the BioBrick Assembly Method was used to combine the promoters with GFP. Subsequently, electrocompetent E. cloni cells were transformed, plated on LB agar plates with antibiotic selection markers, illuminated with blue/UV-light to check for the presence of GFP, and thus a functional device.
For the fluorescent measurements, liquid cultures (3 mL-LB + Antibiotic) were inoculated in polypropylene round-bottom tubes and incubated for 16 to 18 hours in a shaking incubator (200 rpm) at 37 °C. The fluorescence data from cells grown to an OD of ~0.5 (if the OD was higher, it was brought in the range 0.48-0.52) were measured at 300 nm. Finally, the fluorescence data were collected from the overnight cultures of the constructed devices with excitation and emission wavelengths of 483 nm and 525 nm respectively in a 96-well plate by a Tecan Safire2 monochromator MTP Reader. Besides, the absorbance measurements at 600 nm were repeated in the plate reader to normalize for cell density.

Methodology

Preparing electrocompetent cells

- Make a liquid culture of a single colony in 1-3 mL salt free LB
- Grow 300-400 mL cells (without salt) at 37 °C until the O.D. reaches 0.6
- Cool down on ice and perform all the steps at 4 °C
- Spin the cells down in falcon tubes (3500 g, 20 min, 4 °C)
- Resuspend the cells in 10% glycerol, spin the cells down (5000 g, 10 min, 4 °C). Repeat this step 3 times
- Resuspend the cells in 10% glycerol to obtain a dense pulp (usually not more than 1.5 mL)
- Take 50 µL sample and do the electroporation test (without DNA). Pulses should be between 4 and 6 msec. If shorter, wash the cells once again with 30 mL glycerol
- Aliquot the cells (50 µL), quick-freeze in liquid nitrogen and store at -80 °C

Electroporation

- Add 1 µl DNA to 50 µl electrocompetent cells in an ice-cold cuvette (1 mm)
- Electroporate (Eppendorf, 1700 V, 4 msec)
- Add 950 µl of SOC solution
- Incubate for one hour at 37 °C
- Plate out on pre-warmed plates containing the correct selective medium, in this case chlormaphenicol for J23101, J23106 and J23117 and ampicillin for I13504 (37 °C)

BioBrick Assembly Method

- Digest I13504 (GFP) with XbaI and PstI in Tango buffer
- Digest the promoters J23101, J23106 and J23117 with PstI in buffer O
- Load the digested I13504 on a 1.5% agarose gel and visualize it under UV light.
- Thereafter, perform a gel purification of I13504 (GeneJET Gel Extraction Kit - - ThermoFisher Scientific)
- PCR purify the promoters J23101, J23106 and J23117
- Digest the promoters J23101, J23106 and J23117 with FD SpeI in 10x Fast Digest Buffer
- Ligate every promoter with I13504 using T4 DNA ligase

Restriction Mapping

- Digest with NcoI (cuts 1x in pSB1C3) and XhoI (cuts 1x in GFP) in Tango buffer
- Mix gently and spin down
- Incubate for 2 hours at 37 °C in a heating block
- Separate the fragments using gel electrophoresis on a 1.5% agarose gel

Worksheet

Our wetlab team worked well together to fulfill this challenge. Vincent Van Deuren and Laurens Vandebroek performed the BioBrick assembly and the transformation experiments. The measurements were recorded by Laetitia Van Wonterghem, Ovia Margaret Thirukkumaran and Laurens Vandebroek. Laura Van Hese, Astrid Deryckere, Ines Cottignie and Vincent Van Deuren carried out the restriction digestion to check for the inserts. Finally, the results were processed by Ovia Margaret Thirukkumaran and Laurens Vandebroek and our wiki-page was filled with provided data by Vincent Van Deuren and Laetitia Van Wonterghem. Our supervisor Katarzyna Malczewska coordinated the overall works and the rest of the team members served with a helping hand whenever needed.

To grow our cells, we made use of a New Brunswick Innova® 43/43R Shaker purchased from Eppendorf. This incubator has a throw of 2.54 cm. Our devices were measured by a Tecan Safire2 monochromator MTP Reader. This machine was last calibrated on the 31th of March in 2015 by Tecan and our measurements took place on the 25th of August in 2015. The cells were excited at 483 nm and the emission was recorded at 525 nm. To capture the light emission, a Quad4 Monochromator was used. The absorbance was measured at 600 nm with a sampling frequency of 0.11 seconds/ sample while the sampling frequency of the fluorescence was 0.15 seconds/sample.

Contact

Address: Celestijnenlaan 200G room 00.08 - 3001 Heverlee
Telephone: +32(0)16 32 73 19
Email: igem@chem.kuleuven.be