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

Revision as of 22:26, 14 September 2015

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main

Research

Interlab

Modeling

Outreach

Future

Team

Notebook

Protocol

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 degrees Celcius. 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) in 37°C until the O.D.reaches 0.6
Cool down on ice and from now on 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). You should have a pulse of 4-6 msec. If it is shorter, wash the cells once again with 30 mL glycerol
Aliquot the cells (50 µL) and 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 this out on pre-warmed plates (37 °C)
J23101, J23106 and J23117 were plated out on chloramphenicol and I13504 was plated out on ampicillin

BioBrick Assembly Method

Digest I13504 (GFP) with XbaI and PstI in the 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 visualise 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 a Tango buffer
Mix gently and spin down
Put this for 2 hours at 37 °C in a heating block
Gel electrophoresis of the samples in 1.5% agarose gel
Analyse the gel picture and interpret the results

Worksheet

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

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.

@@ Line 67: / Line 67: @@
                  </h2>
                  <p>
-                     We began our experiments by constructing devices that contained constitutive
+                     Experiments started with the construction of devices that contained constitutive
                      promoters with low (J23117), medium (J23106) and higher (J23101) strength.
-                     Each promotor was coupled to biobrick I13504, containing a RBS, GFP protein and a double terminator.
+                     Each promoter was coupled to BioBrick I13504, containing a RBS, GFP protein and a double terminator.
-                     We transformed the above mentioned biobrick and the promoters in E.
+                     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
-                    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
-                     marker. As a positive control, we transformed the cells with pUC19 plasmid and
+                     plated on LB plates containing ampicillin. E. cloni without any
-                     plated them on LB plates containing ampicillin. We also plated E. cloni without any
+                     plasmid was also plated as a negative control on LB plates containing chloramphenicol.
-                     plasmid as a negative control on LB plates containing chloramphenicol. We
+                     Transformation of the BioBricks was performed twice by using chemically competent
-                     performed transformation of the biobricks twice by using chemically competent
+                     cells. The first time, no colonies from any of the four BioBricks were obtained. The
-                     cells. The first time, we did not obtain any colonies of the four biobricks. The
+                     second time, only a few colonies grew. Nevertheless, the positive controls were
-                     second time we got very few colonies. 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
-                     correct every time, and we did double check the efficiency of the cells that
+                     colonies grew to perform the measurements.
-                    proved to be very high. We concluded that our constructs were not easy to
-                    transform into our E. cloni. Therefore, we
-                    switched to electroporation. This technique gave a higher efficiency and enough
-                     colonies for our experiments.
                      <br></br>
-                     Thereafter we proceeded using the Biobrick Assembly Method to assemble the DNA.
+                     Thereafter, the BioBrick Assembly Method was used to combine the promoters with GFP.
-                     Subsequently we performed transformation using electrocompetent E.cloni cells,
+                     Subsequently, electrocompetent E. cloni cells were transformed,
-                     plated them in LB agar plates with antibiotic selection markers, and the plates
+                     plated on LB agar plates with antibiotic selection markers, illuminated with blue/UV-light to check for the presence of GFP, and thus
-                    were illuminated with blue/UV-light to check for the presence of GFP, and thus
+                     a functional device.
-                     the functioning device.
                  </br>
-                 For the fluorescent measurements, we inoculated liquid cultures(3
+                 For the fluorescent measurements, liquid cultures (3
-                 mL-LB + Antibiotic) in polypropylene round-bottom tubes and incubated them for 16
+                 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 degrees. We recorded the
+                 to 18 hours in a shaking incubator (200 rpm) at 37 degrees Celcius. The
-                 fluorescent data from cells grown to an OD of ~0.5 (if the OD was higher, it was brought
+                 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) at 300 nm. Finally, the fluorescence data were collected
+                 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 an excitation and
+                 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 an
+                 emission wavelengths of 483 nm and 525 nm respectively in a 96-well plate by a
-                 Tecan Safire2 monochromator MTP Reader. Also, the absorbance measurements at 600
+                 Tecan Safire2 monochromator MTP Reader. Besides, the absorbance measurements at 600
-                 nm were repeated in the plate reader. This is important because the absorbance
+                 nm were repeated in the plate reader to normalize for cell density.
-                depends on the path length.
              </br>
          </p>
@@ Line 151: / Line 145: @@
 <div class="togglebar">
 <div class="togglethree">
-<h2>Biobrick Assembly Method</h2>
+<h2>BioBrick Assembly Method</h2>
 </div>
 <div id="togglethree" >
@@ Line 187: / Line 181: @@
                  </h2>
                  <p>
-                    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 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>
 <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.