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

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Revision as of 15:22, 14 September 2015

Logo

Protocol

Introduction

We began our experiments by constructing 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. We transformed the above mentioned biobrick and the promoters 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, we transformed the cells with pUC19 plasmid and plated them on LB plates containing ampicillin. We also plated E. cloni 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 into our E. cloni. Therefore, we switched to electroporation. This technique gave a higher efficiency and enough colonies for our experiments.

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.
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 was higher, it was brought 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.

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.

Results

Click here to discover our results!

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Contact

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