Team:Kent/InterLab

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Interlab

Introduction

The InterLab Measurement Study was designed to be an international collaborative study with the aim of standardizing fluorescent measurements while increasing the reproducibility of biomedical research resulting data. [1] . Minimizing the effects of environmental factors is always difficult so the InterLab study set out to combat this by concentrating on the instruments used, which in our case was the plate reader. This year, the study was focused on the expression of GFP in E.coli DH5 alpha cells using different ribosome promoters and binding sites.
Each iGEM team worldwide is provided with the same DNA distribution kit plates and therefore all possess the same constructs and protocols. We were all asked to transform our kits into the E.coli DH5 alpha. Although each team is using different plate readers, we were all required to use a 96-well plate to minimize any discrepancies. The aim of such a study is to allow for a better understanding of the effects of variation on laboratory results while identifying such sources to implement their control in increasing reliable reproducibility of measurements, particularly fluorescence in this case.

Parts Used

This years’ study involved the use of two separate ribosome-binding sites that were tested with three different promoter regions alongside controls (both positive and negative). The readings were related based upon the GFP fluorescence readings produced. We followed the iGEM plate reader protocol
(https://static.igem.org/mediawiki/2017/8/85/InterLab_2017_Plate_Reader_Protocol.p df)

Results

Figure 1: Graph showing the absorbance readings of each of our samples. The amount of light absorbed at 600nm is shown here, allowing insight into the density of the cell and therefore the amount of bacteria present at each time point.

Figure 2: Graph showing the fluorescence readings of each of our samples. The amount of light emitted by each of our samples is shown here, representing the amount of GFP present at each time point.

References

[1] J. Beal, T. Haddock-Angelli, M. Gershater, K. d. Mora, M. Lizarazo, J. Hollenhorst, R. Rettberg and i. I. S. Contributors, “Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli, “ PLOS ONE, vol. 11, supb. E0150182, Mar. 2016