Team:Dalhousie Halifax NS/InterLab

Interlab

What is Interlab?


Scientific discovery depends on having consistent results. Without these valid results it becomes difficult to make any real conclusions or progress. One way that variability can arise between teams is through measurements, especially measurements that involve complicated machines and procedures. The InterLab study is a way to test how different measurements can be between teams while performing the same experiment. This year the InterLab study is attempting to minimize variability encountered in fluorescence measurement, by having teams across the world measure the same fluorescing protein: green fluorescent protein (GFP). More specifically, we will attempt to reduce variability in fluorescence measurements among different labs by standardizing to absolute cell count or colony-forming units (CFUs), rather than the practice of measuring absorbance using optical density (OD) units.

Background

Our Experiment Summary
  • We will be performing the same experiment as teams across the world. This year, the Dalhousie iGEM team opted to solely participate in the Plate Reader and CFU Protocol InterLab experiment. Therefore, a plate reader that could measure both fluorescence and absorbance was used, to permit reproducibility. Prior to taking cell measurements, our InterLab team performed three calibration protocols: OD600 reference point, a particle standard curve and fluorescein standard curve. These initial unit calibration measurements are essential to comparison of data among participating teams. Next, over the period of three days, the cell measurement protocol was tackled. This consisted of first, on Day 1, transforming the Eshcerichia coli DH5 alpha with plasmids, all in pSB1C3. Next, on Day 2, LB medium was inoculated with successful colonies from the transformed plates. Finally on Day 3, following cell growth and sampling of cultures, fluorescence measurement and assay of samples was conducted. The same conditions (same plates and volumes) and settings used during the calibration protocols and measurements were similarly applied.

Our Experience

Triumphs and Troubleshooting

    Our InterLab experience was not without its trials and tribulations, as we continually found ourselves obtaining outlier results, outside the scope of the study. On three separate occasions, our InterLab team repeated the calibration protocols and 3-day cell measurement protocols. However, these additional attempts still resulted in fluorescent measurement data outside the expected scope of the study, and therefore did not pass iGEM Measurement Committee validation checks. While our InterLab Forms I, II, III and IV were all submitted and accepted by the committee, our excel sheet detailing our fluorescent standard curves generated and data was not. Our troubleshooting was not fruitless however, as during our run-throughs of the protocols, our team gained valuable insight into this year’s InterLab project’s protocols and methods. Below are the problems we encountered with the protocols during our initial experiment as well as subsequent troubleshooting:

  • Several test devices did not grow as we expected; this was additionally replicated in our second run-through of the experiment


  • During the 3rd run-through of our protocol, our team struggled to get the DNA from the kit plate to transform, therefore we could not proceed with the protocol and attempt future experiment trials

    • The Dalhousie InterLab team is a crucial subset of our Dalhousie iGEM family. We are composed of determined and inspired young scientists. As such, we greatly appreciated the chance, provided by the InterLab Measurement Committee, to contribute our findings and data in order to potentially create a new standard of measuring fluorescence that would reform current (and diverse) standards of measurements used by labs around the world. Although our results did not pass validation checks, our effort poured into this year’s InterLab study was not unnoticed; the Dalhousie iGEM InterLab team’s hard work was considered sufficient for completing the bronze medal criteria!