Team:KU Leuven/InterLabStudy/Protocol

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Interlab

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main

Research

Interlab

Modeling

Outreach

Future

Team

Notebook

Protocol

Introduction

We began our experiments by constructing devices that contained constitutive promoters with low (J23117), medium (J23106) and higher (J23101) levels of GFP expression. Each device contains the biobrick I13504, necessary for GFP expression. We transformed the above mentioned biobrick and the promoters in E. cloni competent cells. The cells were grown on a LB (from Sigma) 1.5% agar (from 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 cells 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 the bacteria. Therefore, to have more effective transformation, 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 is higher bring it 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

1. Make a liquid culture of a single colony in 1-3 mL salt free LB
2. Grow 300-400 mL cells (without salt) in 37°C until the O.D.reaches 0.6
3. Cool down on ice and from now on perform all the steps at 4 °C
4. Spin the cells down in falcon tubes (3500 g, 20 min, 4°C)
5. Resuspend the cells in 10 % glycerol, spin the cells down (5000 g, 10 min, 4 °C). Repeat this step 3 times
6. Resuspend the cells in 10 % glycerol to obtain a dense pulp (usually not more than 1.5 mL)
7. 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
8. Aliquot the cells (50 µL) and quick-freeze in liquid nitrogen and store at -80 °C