Difference between revisions of "Team:UIUC Illinois/Interlab Study"

The focus of the 2017 Interlab Study was on establishing a standard GFP measurement protocol by applying engineering principles. The protocol is designed such that any lab or individual owning a plate reader could obtain comparable GFP measurements that can be assigned absolute units as opposed to being described in relative levels. We recognize the importance of this study because a major challenge in synthetic biology is the lack of a standard fluorescence measurement protocol and the use of common, comparable units for fluorescence.

This year’s Interlab Study directly addresses the one of the sources of imprecision indicated in the paper published in PLOS using data from the 2016 Interlab Study [1]. A source of imprecision identified while analyzing last year’s data was the lack of use of standard protocols across teams for usage of instruments and analysis of data.

The protocols used for the 2017 Interlab Study thus contained a checklist format, achieved through the pdf and google form format of the protocols, for teams to indicate completion of the protocol. This ensured that the protocol was communicated clearly and that the protocol was followed to a higher degree of accuracy. This year we are participating in the Interlab Study to help answer the following question: How close can the numbers be when fluorescence is measured around the world?

This year, participating teams from around the world all performed the same experiments using the protocols provided by iGEM and the RBS devices provided by iGEM for testing. These RBS devices are expected to allow for more precise and reliable gene expression. All teams used the same constructs, protocols, and chassis. The equipment each team used depended on the availability of either a flow cytometer or a plate reader.

Access to the flow cytometer in our lab, housed under the Institute for Genomic Biology (IGB), is out-of-bounds to undergraduates, which prevented us from carrying out the Flow Cytometer Protocol. Hence, we contributed to the study using Plate Reader Protocol. IGEM HQ provided us with the constructs and control devices on which we performed the measurement protocol, and a spreadsheet through which we submitted our data.

For all plate reading measurements, we used the Tecan Infinite M1000 Pro. 

Figure 1. The above fluorescein standard curve was constructed using the fluorescein measurements made from the TecanM1000 Pro. To achieve the linearity of the log scale standard curve, the fluorescein measurements at 50µM and 25µM were omitted. Although the optimal gain of the Tecan M1000 Pro was determined to be 62, the standard curve did not show a 1:1 slope behavior using the data points at 50µM and 25µM.

Access to our data sheet submitted for the interlab study can be found here (https://static.igem.org/mediawiki/2017/f/f5/UIUC_data.pdf).

References
1) Beal J, Haddock-Angelli T, Gershater M, de Mora K, Lizarazo M, Hollenhorst J, Rettbug R, iGEM Interlab Study Contributers. (2016) Correction: Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli. PLoS ONE 11(3): e0150182. pmid:26937966