Difference between revisions of "Team:KU Leuven/InterLabStudy/Protocol"
| Line 70: | Line 70: | ||
promoters with low (J23117), medium (J23106) and higher (J23101) strength. | 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. | 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 | + | 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 |
marker. As a positive control, cells were also transformed with the pUC19 plasmid and | marker. As a positive control, cells were also transformed with the pUC19 plasmid and | ||
| − | plated on LB plates containing ampicillin. E. cloni without any | + | plated on LB plates containing ampicillin. <i>E. cloni</i> without any |
plasmid was also plated as a negative control on LB plates containing chloramphenicol. | plasmid was also plated as a negative control on LB plates containing chloramphenicol. | ||
Transformation of the BioBricks was performed twice by using chemically competent | 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 | 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 | 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 | + | 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. | colonies grew to perform the measurements. | ||
<br></br> | <br></br> | ||
Thereafter, the BioBrick Assembly Method was used to combine the promoters with GFP. | Thereafter, the BioBrick Assembly Method was used to combine the promoters with GFP. | ||
| − | Subsequently, electrocompetent E. cloni cells were transformed, | + | 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 | 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. | a functional device. | ||
| Line 88: | Line 88: | ||
</br> | </br> | ||
| − | For the fluorescent measurements, liquid cultures (3 | + | 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 | |
| − | to 18 hours in a shaking incubator (200 rpm) at 37 | + | |
fluorescence 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) were measured 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 | ||
| Line 112: | Line 111: | ||
<p>Make a liquid culture of a single colony in 1-3 mL salt free LB | <p>Make a liquid culture of a single colony in 1-3 mL salt free LB | ||
<br/> | <br/> | ||
| − | Grow 300-400 mL cells (without salt) at | + | Grow 300-400 mL cells (without salt) at 37 °C until the O.D. reaches 0.6</br> |
| − | Cool down on ice and perform all the steps at | + | Cool down on ice and perform all the steps at 4 °C</br> |
| − | Spin the cells down in falcon tubes (3500 g, 20 min, | + | Spin the cells down in falcon tubes (3500 g, 20 min, 4 °C)</br> |
| − | Resuspend the cells in 10% glycerol, spin the cells down (5000 g, 10 min, 4 | + | Resuspend the cells in 10% glycerol, spin the cells down (5000 g, 10 min, 4 °C). Repeat this step 3 times</br> |
| − | °C). Repeat this step 3 times</br> | + | |
Resuspend the cells in 10% glycerol to obtain a dense pulp (usually not more | Resuspend the cells in 10% glycerol to obtain a dense pulp (usually not more | ||
than 1.5 mL)</br> | than 1.5 mL)</br> | ||
| Line 122: | Line 120: | ||
between 4 and 6 msec. If shorter, wash the cells once again with 30 mL | between 4 and 6 msec. If shorter, wash the cells once again with 30 mL | ||
glycerol</br> | glycerol</br> | ||
| − | Aliquot the cells (50 µL), quick-freeze in liquid nitrogen and store at - | + | Aliquot the cells (50 µL), quick-freeze in liquid nitrogen and store at -80 °C</br></p> |
</div> | </div> | ||
</div> | </div> | ||
| Line 135: | Line 133: | ||
Electroporate (Eppendorf, 1700 V, 4 msec)</br> | Electroporate (Eppendorf, 1700 V, 4 msec)</br> | ||
Add 950 µl of SOC solution</br> | Add 950 µl of SOC solution</br> | ||
| − | Incubate for one hour at | + | Incubate for one hour at 37 °C</br> |
| − | Plate out on pre-warmed plates containing the correct selective medium, in this case chlormaphenicol for J23101, J23106 and J23117 and ampicillin for I13504 ( | + | 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)</br></p> |
</div> | </div> | ||
</div> | </div> | ||
| Line 165: | Line 163: | ||
<p>Digest with NcoI (cuts 1x in pSB1C3) and XhoI (cuts 1x in GFP) in Tango buffer</br> | <p>Digest with NcoI (cuts 1x in pSB1C3) and XhoI (cuts 1x in GFP) in Tango buffer</br> | ||
Mix gently and spin down</br> | Mix gently and spin down</br> | ||
| − | Incubate for 2 hours at | + | Incubate for 2 hours at 37 °C in a heating block</br> |
Separate the fragments using gel electrophoresis on a 1.5% agarose gel</br></p> | Separate the fragments using gel electrophoresis on a 1.5% agarose gel</br></p> | ||
</div> | </div> | ||
Revision as of 19:30, 15 September 2015
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 °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
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 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
