Fifth International InterLab Measurement Study
Please note: this is an optional and voluntary exercise for all teams. This is also an optional activity for fulfilling the Bronze Medal Requirement #4 this year.
All of the 2018 iGEM teams are invited and encouraged to participate in the Fifth International InterLaboratory Measurement Study in synthetic biology. We’re hoping this study will get you excited for iGEM and help prepare you for the rest of your project!
Introduction to the InterLab Study
Reliable and repeatable measurement is a key component to all engineering disciplines. The same holds true for synthetic biology, which has also been called engineering biology. However, the ability to repeat measurements in different labs has been difficult. The Measurement Committee, through the InterLab study, has been developing a robust measurement procedure for green fluorescent protein (GFP) over the last several years. We chose GFP as the measurement marker for this study since it's one of the most used markers in synthetic biology and, as a result, most laboratories are equipped to measure this protein.
We aim to improve the measurement tools available to both the iGEM community and the synthetic biology community as a whole. One of the big challenges in synthetic biology measurement has been that fluorescence data usually cannot be compared because it has been reported in different units or because different groups process data in different ways. Many have tried to work around this using “relative expression” comparisons; however, being unable to directly compare measurements makes it harder to debug engineered biological constructs, harder to effectively share constructs between labs, and harder even to just interpret your experimental controls.
The InterLab protocol aims to address these issues by providing researchers with a detailed protocol and data analysis form that yields absolute units for measurements of GFP in a plate reader.
A Brief History of the InterLab
Over the past four years, iGEM has advanced the frontiers of science with the biggest interlaboratory studies ever done in synthetic biology. These studies established a baseline for replicability of fluorescence measurements and identified likely key sources of error, and have now been published as an open-access journal article in PLOS ONE.
To read the article on the 2014 and 2015 studies, go to the following URL: http://dx.doi.org/10.1371/journal.pone.0150182.
A publication on the 2016 interlab study is currently under review and a publication on the 2017 interlab study is in preparation. Preliminary analyses of both of these were presented at iGEM and are available online: iGEM 2016 Interlab Results Presentation, iGEM 2017 Interlab Results Presentation.
Goal for the Fifth InterLab
The goal of the iGEM InterLab Study is 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. Until we reach this point, synthetic biology will not be able to achieve its full potential as an engineering discipline, as labs will not be able to reliably build upon others’ work.
In the previous studies, we showed that by measuring GFP expression in absolute fluorescence units calibrated against a known concentration of fluorescent molecule, we can greatly reduce the variability in measurements between labs. However, when we take bulk measurements of a population of cells (such as with a plate reader), there is still a large source of variability in these measurements: the number of cells in the sample.
Because the fluorescence value measured by a plate reader is an aggregate measurement of an entire population of cells, we need to divide the total fluorescence by the number of cells in order to determine the mean expression level of GFP per cell. Usually we do this by measuring the absorbance of light at 600nm, from which we compute the “optical density (OD)” of the sample as an approximation of the number of cells. OD measurements are subject to high variability between labs, however, and it is unclear how good of an approximation an OD measurement actually is. If we used a more direct method to determine the cell count in each sample, then potentially we could remove another source of variability in our measurements.
This year, we want your help in answering the following question: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?
In order to compute the cell count in your samples, we will have you use two orthogonal approaches:
1. Converting between absorbance of cells to absorbance of a known concentration of beads.
- Absorbance measurements use the way that a sample of cells in liquid scatter light in order to approximate the concentration of cells in the sample. In this year’s Measurement Kit, we provide you with a sample containing silica beads that are roughly the same size and shape as a typical E. coli cell, so that it should scatter light in a similar way. Because we know the concentration of the beads, we can convert each lab’s absorbance measurements into a universal, standard “equivalent concentration of beads” measurement.
2. Counting colony-forming units (CFUs) from the sample.
- A simple way to determine the number of cells in a sample of liquid media is to pour some out on a plate and see how many colonies grow on the plate. Since each colony begins as a single cell (for cells that do not stick together), we can determine how many live cells were in the volume of media that we plated out and obtain a cell concentration for our sample as a whole. We will have you determine the number of CFUs in positive and negative control samples in order to compute a conversion factor from absorbance to CFU.
By using these two approaches, we will be able to determine how much they agree with each other, and whether using one (or both) can help to reduce lab-to-lab variability in measurements. If it can, then together we will have brought synthetic biology one step closer to becoming a true, reliable engineering discipline. So, can you measure GFP fluorescence in a plate reader? Does working on an international, collaborative experiment sound exciting? Then this is the perfect study for you!
Requirements for successful completion of the InterLab Study
These guidelines are intended to make sure that each submitted data set follows protocol and measurement instructions well enough to be comparable to other interlab data, and thus contribute to the collective goals of the interlab study. Therefore, all teams who wish to use the Interlab study to fulfill the bronze medal requirements must satisfy these requirements:
- 1. Measure both absorbance and fluorescence on a plate reader. If you do not have a plate reader capable of recording both, we suggest that you collaborate with another team.
- 2. Use E. coli strain DH5-alpha for all cell measurements. If you do not have this strain available, please collaborate with another team to obtain it.
- 3. Your data must pass “common sense” validity checks, including: dilution makes absorbance and fluorescence values go down, positive control is brighter than negative control, cells grow over time, test devices aren’t all the same value. We expect you to check your data before submission.
- 4. All forms and data must be received by the Measurement Committee on time. Late submissions and missed deadlines will not be accepted.
- 5. You must edit your team's InterLab page (ex: http://2018.igem.org/TeamName:Example/InterLab) and add a summary of the work you conducted. You must remove the Alert box from the top of the page.
- May 1: Registration for the Interlab Study opens (see InterLab Sign Up button at top of page).
- June 29: Registration for the Interlab Study ends. Teams must register within this time window in order to use the Interlab Study to fulfill their bronze medal requirements.
- July 27: Data (in Excel sheets) and completion of the four online forms are due.
- July 30: The Measurement Committee will review data sets for validity. Teams will be notified if their data sets are valid by July 30th. If your team’s data set is acceptable, no further action will be required.
- August 24: If you are notified that your data set is not valid, you will be permitted to repeat your measurements and resubmit your data set. The Measurement Committee will explain why your data was not valid in order to help you obtain valid data. Resubmissions are due by August 24th, 2018.
Send questions to the Measurement Committee (measurement [AT] igem [DOT] org). Please also contact the Measurement Committee well ahead of time if you need to make alternative arrangements for submission due to internet limitations in your country.
Good luck, and thank you for participating in the Interlab Study!