Team:Oxford/InterlabMeasurement

Time-course Protocol
To understand the growth of the transformed DH5-a, we measured the Optical Density (OD), and corresponding Fluorescence (Fluo) for each device over the course of 40 cycles. This series of readings took over 13 hours, fully capturing the entire growth curve of the bacteria. 3 cultures for each device were set-up overnight, taken from separate single colonies on the agar plate (biological replicates), as well as 3 cultures of non-transformed DH5-a (negative control). Of these 12 cultures, 3 repeated samples were taken to be read in our laboratory’s plate-reader. We used an Infinite m2000 plate reader, with a Greiner 96 Flatback plate across all protocol. The resulting growth curves showed no peculiarities in either measurement, leading us to then decide to take single-readings of each culture at, or close to, the end of the log-phase.

Single-readings
As before, 12 cultures were set up overnight. In the morning, 10ul were transferred from the overnight cultures into 5ml of LB broth, with appropriate amounts of antibiotic dependent on the device being used. These cultures were incubated and shaken at 37°C until their OD reached, or was close to, 0.6. This took approximately 3 hours. After this time, 1ml of each culture was centrifuged for 2 minutes, pipetting out all the supernatant. To re-suspend the pellet, we used 1X M9 salts so as to minimise autofluorescence contributing to our final readings. The amount of minimal media used for each culture was different dependent on the respective ODs. This was to standardise the amount of cells being used in the plate-reader. The specific volume of M9 salts was determined by (OD/0.6)ml. For each culture, 200ul were pipetted into the Greiner plate. This was repeated twice, giving two technical replicates and 24 wells occupied with samples. In addition, two wells were filled with M9 salts, as negative controls. Leaving the cover off, the plate was read once using the following settings: Excitation 485nm, Emission 510nm Shaking before – 30 seconds Gain 2000

This protocol was repeated the following day, using new individual colonies from the original agar plates.

We also took epifluorescent images of each cultures after the plate-readings, to visually inspect the variation in fluorescence per cell, and confirm that what we saw matched the data we were given by the plate-reader. For this we used a Nikon TE-200 microscope with GFP Filter set (Chroma), Hamamatsi EMCCD Camera and simple PCI software.