Team:Thessaloniki/InterLab

Interlab

Interlab

Our Team participated in iGEMs fifth Interlaboratory Study, a study organized by IGEM Measurement Committee in order to identify and correct the sources of systematic variability in synthetic biology measurements, so that eventually, measurements that are taken in different labs will be no more variable than measurements taken within the same lab. This year’s InterLab study aims to establish a GFP measurement protocol based on engineering principles that anyone with a plate reader can use in their lab and normalize fluorescence measurements to absolute cell count or colony forming units (CFUs).

To begin with our experiments we needed a plate reader that is qualified to measure GFP fluorescence and OD600. In our city, there was no such an instrument so we travelled to Alexandroupoli, at the Democritus University of Thrace, where we used the Perkin Elmer EnSpine Multimode Plate Reader. For all of the callibration protocols and cell measurements we adjusted the exitation and emission settings to 485nm and 520nm respectively, the number of flashes used was 10 and black plates with clear flat bottom were used.

Calibration Protocols:

Before starting the plate reader measurements , we obtained the OD600 reference point, the particle standard curve and then the fluorescein standard curve.

The OD600 reference point was calculated by using LUDOX CL-X suspension to obtain a conversion factor to transform Absorbance (Abs600) data from the plate reader into a comparable OD600 measurements that would be obtained in a spectrophotometer.

Then, we obtained the particle standard curve by preparing a dilution series of mono disperse silica microspheres and measuring the Abs600 in the plate reader.

The last calibration protocol was conducted to generate the fluorescence standard curve by preparing a dilution series of fluorescein and then measuring the fluorescence in the plate reader. Fluorescein is a small molecule which has similar excitation and emission properties to GFP, but also cost effective and easy to handle.

The standard curve slopes showed a somewhat polynomial shape although they were supposed to have a 1:1 slope. This could be due to a consistent pipetting error or because of the over saturation of the detector.

Cell Measurement:

The following devices were characterised:

• six test devices encoding GFP fused with different promoters and RBSs.
  BBa_J364000, BBa_J364001, BBa_J364002, BBa_J364007, BBa_J364008, BBa_J364009
• a positive control BBa_I20270
• a negative control BBa_R0040

Each one of the 6 test devices has a different Anderson promoter. So, we are expecting different fluorescence levels for each device depending on the Anderson promoter used.

Transformation of 8 test devices into competent DH5α cells:

All the plasmids were successfully transformed into DH5a chemically competent cells and resulted in a sufficient amount of colonies on all plates. Then, two colonies from each plate were picked and inoculated overnight (14-16 hours) at 37°C and 220 rpm.

Fluorescence and OD600 Measurement for the Transformed Parts:

The OD600 and the fluorescence of the transformed cells were measured according to the protocol after 0 and 6 hours. Measurements of both of these time points gave us the following data

Discussion:

Devices 1 and 5 did not grow until the 6 hour timepoint, so the resulted fluorescence is not representative of the promoters strenght. In addition, Device 4, which had the strongest Anderson Promoter, did not grow as much as the other Devices but still the Fluorescence was high. All the other devices grew normally and the results we aqcuired were the ones expected. Conclusively, we completed our Cell Measurement Protocol with success.

CFU (Colony Forming Units)

This year, for the interlaboratory study, teams measure CFU/ml/0.1OD600 in addition to the standard Plate Reader fluorescence measurements as was the case in previous years. Here, we document the results of our CFU measurements. The protocol proposed at pages 13-14 of the interlab .pdf file provided in the interlab page at igem.org, “Protocol: Colony Forming Units per 0.1 OD600 E. coli cultures” was faithfully followed.

This year, for the interlaboratory study, teams measure CFU/ml/0.1OD600 in addition to the standard Plate Reader fluorescence measurements as was the case in previous years. Here, we document the results of our CFU measurements. The protocol proposed at pages 13-14 of the interlab .pdf file provided in the interlab page at igem.org, “Protocol: Colony Forming Units per 0.1 OD600 E. coli cultures” was faithfully followed.

The Transformations were performed on Frozen Competent DH5α cells (7/12). The CFU process (dilutions and plating) took place on 14th of July. The next day colonies were counted. All protocols followed were provided by iGEM.