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

 
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                     <h2>
 
                     <h2>
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                         Protocol
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                         Protocols
 
                     </h2>
 
                     </h2>
 
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                 </h2>
 
                 </h2>
 
                 <p>
 
                 <p>
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                     We began our experiments by constructing devices that contained constitutive
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                     Experiments started with the construction of devices that contained constitutive
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                     promoters with low (J23117), medium (J23106) and higher (J23101) levels of GFP
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                     promoters with low (J23117), medium (J23106) and higher (J23101) strength.
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                     expression. Each device contains the biobrick I13504, necessary for GFP
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                     Each promoter was coupled to BioBrick I13504, containing a RBS, GFP protein and a double terminator.
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                     expression. We transformed the above mentioned biobrick and the promoters in E.
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                     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
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                    cloni competent cells. The cells were grown on a LB (from Sigma) 1.5% agar (from
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                     marker. As a positive control, cells were also transformed with the pUC19 plasmid and
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                    VWR Chemicals) plates with chloramphenicol (from Acros Organics) as a selection
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                     plated on LB plates containing ampicillin. <i>E. cloni</i> without any
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                     marker. As a positive control, we transformed the cells with pUC19 plasmid and
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                     plasmid was also plated as a negative control on LB plates containing chloramphenicol.  
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                     plated them on LB plates containing ampicillin. We also plated cells without any
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                     Transformation of the BioBricks was performed twice by using chemically competent
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                     plasmid as a negative control on LB plates containing chloramphenicol. We
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                     cells. The first time, no colonies from any of the four BioBricks were obtained. The
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                     performed transformation of the biobricks twice by using chemically competent
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                     second time, only a few colonies grew. Nevertheless, the positive controls were
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                     cells. The first time, we did not obtain any colonies of the four biobricks. The
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                     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
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                     second time we got very few colonies. Nevertheless, the positive controls were
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                     colonies grew to perform the measurements.
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                     correct every time, and we did double check the efficiency of the cells that
+
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                    proved to be very high. We concluded that our constructs were not easy to
+
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                    transform the bacteria. Therefore, to have more effective transformation, we
+
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                    switched to electroporation. This technique gave a higher efficiency and enough
+
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                     colonies for our experiments.
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                     <br></br>
 
                     <br></br>
  
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                     Thereafter we proceeded using the Biobrick Assembly Method to assemble the DNA.
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                     Thereafter, the BioBrick Assembly Method was used to combine the promoters with GFP.
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                     Subsequently we performed transformation using electrocompetent E.cloni cells,
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                     Subsequently, electrocompetent <i>E. cloni</i> cells were transformed,
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                     plated them in LB agar plates with antibiotic selection markers, and the plates
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                     plated on LB agar plates with antibiotic selection markers, illuminated with blue/UV-light to check for the presence of GFP, and thus
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                    were illuminated with blue/UV-light to check for the presence of GFP, and thus
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                     a functional device.
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                     the functioning device.
+
  
 
                 </br>
 
                 </br>
  
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                 For the fluorescent measurements we inoculated liquid cultures(3
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                 For the fluorescent measurements, liquid cultures (3 mL-LB + Antibiotic) were inoculated in polypropylene round-bottom tubes and incubated for 16
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                mL-LB+Antibiotic) in polypropylene round-bottom tubes and incubated them for 16
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                 to 18 hours in a shaking incubator (200 rpm) at 37 °C. The
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                 to 18 hours in a shaking incubator (200 rpm) at 37 degrees. We recorded the
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                 fluorescence data from cells grown to an OD of ~0.5 (if the OD was higher, it was brought
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                 fluorescent data from cells grown to an OD of ~0.5 (if the OD is higher bring it
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                 in the range 0.48-0.52) were measured at 300 nm. Finally, the fluorescence data were collected
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                 in the range 0.48-0.52) at 300 nm. Finally, the fluorescence data were collected
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                 from the overnight cultures of the constructed devices with excitation and
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                 from the overnight cultures of the constructed devices with an excitation and
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                 emission wavelengths of 483 nm and 525 nm respectively in a 96-well plate by a
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                 emission wavelengths of 483 nm and 525 nm respectively, in a 96-well plate by an
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                 Tecan Safire2 monochromator MTP Reader. Besides, the absorbance measurements at 600
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                 Tecan Safire2 monochromator MTP Reader. Also, the absorbance measurements at 600
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                 nm were repeated in the plate reader to normalize for cell density.  
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                 nm were repeated in the plate reader. This is important because the absorbance
+
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                depends on the path length.
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             </br>
 
             </br>
 
         </p>
 
         </p>
 
     </div>
 
     </div>
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    <div class="part">
  
 
         <h2>
 
         <h2>
 
             Methodology</h2>
 
             Methodology</h2>
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             <p>Make a liquid culture of a single colony in 1-3 mL salt free LB
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             <dl>
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        <br/>
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              <dd>- Make a liquid culture of a single colony in 1-3 mL salt free LB</dd>
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         Grow 300-400 mL cells (without salt) in 37°C until the O.D.reaches 0.6</br>
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         <dd>- Grow 300-400 mL cells (without salt) at 37 °C until the O.D. reaches 0.6</dd>
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    Cool down on ice and from now on perform all the steps at 4 °C</br>
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  <dd>- Cool down on ice and perform all the steps at 4 °C</dd>
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Spin the cells down in falcon tubes (3500 g, 20 min, 4°C)</br>
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<dd>- Spin the cells down in falcon tubes (3500 g, 20 min, 4 °C)</dd>
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Resuspend the cells in 10 % glycerol, spin the cells down (5000 g, 10 min, 4
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<dd>- Resuspend the cells in 10% glycerol, spin the cells down (5000 g, 10 min, 4 °C). Repeat this step 3 times</dd>
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°C). Repeat this step 3 times</br>
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<dd>- Resuspend the cells in 10% glycerol to obtain a dense pulp (usually not more
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Resuspend the cells in 10 % glycerol to obtain a dense pulp (usually not more
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than 1.5 mL)</dd>
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than 1.5 mL)</br>
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<dd>- Take 50 µL sample and do the electroporation test (without DNA). Pulses should be
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Take 50 µL sample and do the electroporation test (without DNA). You should have
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between 4 and 6 msec. If shorter, wash the cells once again with 30 mL
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a pulse of 4-6 msec. If it is shorter, wash the cells once again with 30 mL
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glycerol</dd>
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glycerol</br>
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<dd>- Aliquot the cells (50 µL), quick-freeze in liquid nitrogen and store at -80 °C</dd>
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Aliquot the cells (50 µL) and quick-freeze in liquid nitrogen and store at -80
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</dl>
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<div id="toggletwo" >
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<p>Add 1 µl DNA to 50 µl electrocompetent cells in an ice-cold cuvette (1 mm)</br>
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<dl>
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Electroporate (Eppendorf, 1700 V, 4 msec)</br>
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<dd>- Add 1 µl DNA to 50 µl electrocompetent cells in an ice-cold cuvette (1 mm)</dd>
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Add 950 µl of SOC solution</br>
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<dd>- Electroporate (Eppendorf, 1700 V, 4 msec)</dd>
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Incubate for one hour at 37 °C</br>
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<dd>- Add 950 µl of SOC solution</dd>
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Plate this out on pre-warmed plates (37 °C)</br>
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<dd>- Incubate for one hour at 37 °C</dd>
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J23101, J23106 and J23117 were plated out on chloramphenicol and I13504 was
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<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>
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plated out on ampicillin</br></p>
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<h2>Biobrick Assembly Method</h2>
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<h2>BioBrick Assembly Method</h2>
 
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<div id="togglethree" >
 
<div id="togglethree" >
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<p>Digest I13504 (GFP) with XbaI and PstI in the Tango buffer</br>
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<dl>
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Digest the promoters J23101, J23106 and J23117 with PstI in buffer O</br>
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<dd>- Digest I13504 (GFP) with XbaI and PstI in Tango buffer</dd>
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Load the digested I13504 on a 1.5% agarose gel and visualise it under UV light.
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<dd>- Digest the promoters J23101, J23106 and J23117 with PstI in buffer O</dd>
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Thereafter perform a gel purification of I13504 (GeneJET Gel Extraction Kit -
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<dd>- Load the digested I13504 on a 1.5% agarose gel and visualize it under UV light.</dd>
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ThermoFisher Scientific)</br>
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<dd>- Thereafter, perform a gel purification of I13504 (GeneJET Gel Extraction Kit -
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PCR purify the promoters J23101, J23106 and J23117</br>
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- ThermoFisher Scientific)</dd>
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Digest the promoters J23101, J23106 and J23117 with FD SpeI in 10x Fast Digest
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<dd>- PCR purify the promoters J23101, J23106 and J23117</dd>
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Buffer</br>
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<dd>- Digest the promoters J23101, J23106 and J23117 with FD SpeI in 10x Fast Digest
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Ligate every promoter with I13504 using T4 DNA ligase</br></p>
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Buffer</dd>
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<dd>- Ligate every promoter with I13504 using T4 DNA ligase</dd>
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<h2>Restriction mapping</h2>
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<h2>Restriction Mapping</h2>
 
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<p>Digest with NcoI (cuts 1x in pSB1C3) and XhoI (cuts 1x in GFP) in a Tango buffer</br>
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<dl>
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Mix gently and spin down</br>
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<dd>- Digest with NcoI (cuts 1x in pSB1C3) and XhoI (cuts 1x in GFP) in Tango buffer</dd>
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Put this for 2 hours at 37 °C in a heating block</br>
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<dd>- Mix gently and spin down</dd>
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Gel electrophoresis of the samples in 1.5% agarose gel</br>
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<dd>- Incubate for 2 hours at 37 °C in a heating block</dd>
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Analyse the gel picture and interpret the results</br></p>
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<dd>- Separate the fragments using gel electrophoresis on a 1.5% agarose gel</dd>
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</dl>
 
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<h2>This is example five</h2>
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<p>tiralalalala
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<br/>
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tiralalala
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<br/>
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tiralalala<br/></p>
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<div class="part">
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                 <h2>
 
                 <h2>
 
                     Worksheet
 
                     Worksheet
 
                 </h2>
 
                 </h2>
 
                 <p>
 
                 <p>
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                   Our wetlab team worked well together to fulfill this challenge. Vincent Van Deuren and Laurens Vandenbroek performed the biobrick assembly and the transformation experiments. The measurements were recorded by Laetitia Van Wonterghem, Ovia Margaret Thirukkumaran and Laurens Vandenbroek. 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 Vandenbroek, and our wiki-page was filled with provided data by Vincent Van Deuren </br>
+
                   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 in 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.
+
<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.
  
 
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<a href="https://2015.igem.org/Team:KU_Leuven/Research/Idea">
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<a href="https://2015.igem.org/Team:KU_Leuven/InterLabStudy/Results">
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  <h2>Idea</h2>
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  <h2>Results</h2>
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<p> A detailed description about the interaction between our two cells and the genetic circuit can be found here.
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<p>Click here to discover our results.</p>
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<h2>Parts</h2>
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  <h2>Back</h2>
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<p> A list of all used biobricks, the modifications we performed on them and the new ones we designed.
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  <p>Go back to the Interlab page.</p>
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<br/>
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<p> More coming soon
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  <h2>Methods</h2>
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  <p> Here you can find the performed steps to create two different cell types and detailed quantification methods to determine interesting parameters.
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<p> More coming soon
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<p> The results of our experiments will appear here.
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Latest revision as of 11:24, 10 November 2015

Logo

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.

Results

Click here to discover our results.

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Read more
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Results

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Back

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Contact

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