Difference between revisions of "Team:Manchester/InterLab"

Aseptic technique was maintained at all times.

Transformation

The E.coli strain used was Dh5α. The DNA provided in the distribution kit for the transformations was re-suspended using 10 μl of dH2O. Competent cells were transformed using the following 8 devices, inside the plasmid backbone pSB1C3:

- BBa_R0040 (Negative Control)

- BBa_I20270 (Positive Control)

- BBa_J364000 (Device 1)

- BBa_J364001 (Device 2)

- BBa_J364002 (Device 3)

- BBa_J364003 (Device 4)

- BBa_J364004 (Device 5)

- BBa_J364005 (Device 6)

Transformations were done using our refined protocol (see additional work) and the transformed cells were subsequently plated on 8 LB agar plates containing chloramphenicol at a concentration of 25 mg/ml. The plates were incubated overnight at 37°C.

Overnight Cultures

5 ml of sterile LB broth was transferred to a total of 16 x 50 ml falcon tubes; chloramphenicol was then added to each tube at a concentration of 25 mg/ml. Two colonies were picked from each plate, each individual colony was transferred to a separate falcon tube. The tubes were incubated overnight at 37°C and 180 rpm.

Plate Reader Settings

Measurements of optical density at 600 nm (OD600) and fluorescence were taken using a BMG LabTech Clariostar plate reader, provided to us by the Manchester Institute of Biotechnology (MIB). OD600 measurements were all taken at 24°C with 35 flashes, and an orbital averaging of 3. Fluorescence measurements were taken with 8 flashes and a gain of 748. Excitation and emission wavelengths were 515-20 nm and 470-15 nm respectively. Pathlength correction was turned off for both measurements.

OD600 Reference Point

LUDOX S-40 was used as a single reference point to calibrate the plate reader. A ratiometric conversion factor was obtained to ensure that all Absorbance measurements at 600 nm (Abs600) were converted to OD600 measurements, whilst also taking into account differences in instruments.

100 µl of LUDOX was transferred into wells A1-D1 of a black, clear-bottom 96-well plate. 100 µl of distilled H₂O was then added into wells A2-D2. Abs600 measurements of LUDOX and H₂0 were then taken. A ratiometric conversion factor of roughly 3.269 was obtained.

Fluorescence Standard Curve

The fluorescein stock was spun down for 30 seconds at 3000 rpm and then re-suspended in 1 mL of 1x PBS to produce a 2x stock solution (100 μM). This was then further diluted with 1x PBS to a 1x stock solution with a final concentration of 50 μM.

200 µl of the fluorescein stock was transferred into wells A1-D1. The fluorescein stock was then serially diluted in four replicates (A2-A12, B2-B12, C2-C12, D2-D12) by mixing with 1x PBS to obtain 100 µl of 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625, 0.1953125, 0.09765625, 0.048828125, and 0 µM of fluorescein solution. Fluorescence was then measured using a BMG LabTech ClarioStar plate reader, and a fluorescence standard curve generated (Figure 1). This standard curve was used to correct cell based readings to an equivalent fluorescein concentration and measure the concentration of GFP.

Cell Measurements

Overnight cultures of each device were diluted to a target OD of 0.02 and incubated at 37°C and 220 rpm, falcon tubes were wrapped in tin foil throughout. 500 μL of each device was transferred to an Eppendorf tube and put on ice at time points 0 h, 2 h, 4 h, and 6 h. 100 μl of each sample was then transferred to 96 well plate using the provided set-up guidelines and measurements. OD and fluorescence measurements were taken simultaneously.
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