Team:Duesseldorf/InterLab

INTERLAB STUDY

As it was the case the previous four years, iGEM is proposing participation in the worldwide InterLab study again. It is undeniable that scientific exchange represents a huge aspect of innovation and experimental discovery. Thus, to continue the diagnosis of systematic variability, different iGEM teams are encouraged to perform several experiments that will allow an establishment of a standardized GFP measurement protocol, which is applicable everywhere, independent from instruments, materials and procedures used.

Calibrations

Different measurements are performed for ideal calibration conditions. First a measurement with LUDOX CL-X is done, which is a 45% colloidal silica suspension. This allows to get a conversion factor to transform the absorbance (A600) from the plate reader to an OD600 measurement that would be obtained in a spectrophotometer. In addition to that, a serial dilution of monodisperse silica microspheres is performed and Abs600 is determined. Since physical characteristics of these microspheres resemble those of microbial cells, conversion of Abs600 of a known amount of particles per volume can be used to convert this value to a specific number of cells. Finally, since the aim of this study is to create a representative protocol for GFP measurement, a third calibration using fluorescein is done. A calibration using serial dilutions of fluorescein is prepared in order to obtain a final standard fluorescence curve.

Materials
  • Plate reader: TECAN Infinite M200
  • Plate reader plates: black, with clear flat bottom
  • LUDOX CL-X
  • Silica beads
  • Fluorescein

Methods

The experiments were performed according to the iGEM 2018 Interlab study protocol provided by the iGEM headquarter.

Figure 1: Particle standard curve obtained after Abs600 measurement of LUDOX CL-X solution (replicates)
Figure 2: Fluorescence calibration was obtained by measurement of Fluorescein at different concentration after performance of serial dilutions.

Cell experiments

The experiments were performed with the provided protocol. For the measurements the TECAN Infinite M200 was used to measure OD600 and fluorescence.

Materials

  • TECAN Infinite M200
  • 96 well plates (2 in total), black with a flat clear bottom
  • 50 ml Falcon tubes
  • 2 ml tubes
  • 1.5 ml tubes
  • LB medium
  • Chloramphenicol (34 µg/ml)
  • LB agar plates (with chloramphenicol)
  • Incubator

Results

After the transformation we were able to pick two colonies for each construct.

Figure 3: Positive transformants were selected on chloramphenicol plates as described on the interlab study protocol. Grown colonies were picked and grown in liquid media, since the experiments succeeded at the first try, no repetition was necessary.

For the measurements and results of the fluorescence we showed our results in a graph.

Figure 4: In this case not all experiments showed the expected result. The fluorescence in the samples with the devices 4 and 5 showed no increase over 6 hours of incubation. But also in these samples the OD increased over time. The samples were measured as the provided protocol shows. The total values are the average of 4 samples.

The expectations in this case were different:


Device Part Promoter Properties Source
Negative control BBa_R0040 TetR repressible promoter Only with induction with tetracycline, promoter is activated http://parts.igem.org/Part:BBa_R0040
Positive control BBa_I20270 BBa_J23151 Positive control http://parts.igem.org/Part:BBa_I20270
1 BBa_J364000 BBa_J23101 RFU: 1487 http://parts.igem.org/wiki/index.php?title=Part:BBa_J23101
2 BBa_J364001 BBa_J23106 RFU: 1185 http://parts.igem.org/wiki/index.php?title=Part:BBa_J23106
3 BBa_J364002 BBa_J23117 RFU: 162 http://parts.igem.org/wiki/index.php?title=Part:BBa_J23117
4 BBa_J364007 BBa_J23100 RFU: 2547 http://parts.igem.org/wiki/index.php?title=Part:BBa_J23100
5 BBa_J364008 BBa_J23104 RFU: 1831 http://parts.igem.org/wiki/index.php?title=Part:BBa_J23104
6 BBa_J364009 BBa_J23116 RFU: 396 http://parts.igem.org/wiki/index.php?title=Part:BBa_J23116

Experience with the InterLab study

We did not encounter any difficulties performing the InterLab study. The protocols supplied were simple to understand and execute. In order to have iGEM teams in the future profit from our experience with this year’s InterLab challenge, we published the performed experiments on protocols.io. This way we want to contribute to the concept of open science and would like to simplify the performance of these experiments with the easy to use protocols.io app.
We enjoyed participating in this year´s InterLab study as we believe that differences in scientific research and development will be made only if scientists collaborate with each other and work on big projects (such as the Interlab challenge) together. We hope that we will be able to influence and improve the evolving scientific process with our work and are really happy to have been a part of this sensation.