Difference between revisions of "Team:KU Leuven/InterLabStudy/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 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, helped with planning, provided protocols, corrected the input, filled in the required forms and prepared and submitted the descriptions.

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^th of March in 2015 by Tecan and our measurements took place on the 25^th 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.