Fourth International InterLab Measurement Study

Please note: this is an optional and voluntary exercise for all teams. This is also an optional activity for one the Bronze Medal Requirements this year.

All of the 2017 iGEM teams are invited and encouraged to participate in the Fourth International InterLaboratory Measurement Study in synthetic biology. We’re hoping this study will get you excited for iGEM and help prepare you for the summer!

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 three 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 is that fluorescence data usually cannot be compared because it is reported in different units or because different groups process data in different ways.

Often we work around this by doing some sort of “relative expression” comparison; 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 measuring GFP in a plate reader.

A Brief History of the InterLab

Over the past three years, iGEM has advanced the frontiers of science with the two 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, go to the following URL: http://dx.doi.org/10.1371/journal.pone.0150182.

A publication on the 2016 interlab study is currently in preparation.

Results from the 2016 iGEM InterLab Study

Congratulation to all of the 92 teams who participated in this year's interlab study, and who helped contribute to a momentous result: high-precision replication of fluorescence measurements in equivalent units around the world! During iGEM we showed preliminary analysis of the results in the InterLab study workshop, and are posting the slides online here as well for everybody to see: iGEM 2016 Interlab Results Presentation.

Goal for the Fourth InterLab

We want to establish a GFP measurement protocol based on engineering principles that anyone with a plate reader can use in their lab. This means we are looking for teams to use the same exact protocol around the world to produce common, comparable units for measuring GFP with different plate readers.

We have a major question we want to explore with your help: How close can the numbers be when fluorescence is measured all around the world?

This year, we are also going to test some RBS devices (BCDs) that are intended to make gene expression more precise and reliable. How reliable will these devices turn out to be in labs around the world?

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!

InterLab Requirements

In order to participate in this year's study, your team must meet these requirements:

Have access to a plate reader that can read GFP
Competent E. coli DH5-alpha cells
Use the materials iGEM HQ has provided (InterLab Parts and Measurement Kit)
Submit all forms and data by the due date (September 29)
Edit your team's InterLab wiki page (https://2017.igem.org/Team:[TeamName]/InterLab) by the Wiki Freeze (November 1)

Since the InterLab is an optional Bronze Medal activity this year, teams must adhere to these requirements in order to participate. These requirements will also help us make this a more robust engineering standard for measuring GFP in synthetic biology.

2017 InterLab Sign-up Details

Teams need to sign up with the Measurement Committee in order to participate in the InterLab study. It's really easy - just send us an email at measurement AT igem DOT org to sign up!

InterLab Sign up Deadline: ~~July 3, 2017~~ UPDATED DEADLINE: July 9, 2017 11:59pm EST
Please note: This email will reach the entire Measurement Committee. Please wait for either Jake, Markus, or Traci to reply before sending any other emails to iGEM HQ to avoid confusion! It may take us a few days to reply.

Data Submission Deadline: September 29, 2017.
All teams need to fill out the three protocol forms for the plate reader and the Excel data sheets. If you also measured the parts with a flow cytometer, please fill out that form as well.

InterLab Materials

2017 InterLab Parts

This year, we are providing teams with the eight plasmids containing the composite parts they will need for the InterLab Study (details and locations of the parts are listed below in the "Transform the InterLab Parts" section). These plasmids can be found in both Kit Plate 6 and Kit Plate 7 in the 2017 Distribution Kit as dried down DNA samples. (Note: We have sent duplicate samples of the plasmids to ensure all teams can transform the parts needed to participate in the InterLab study. You only need to transform one set of the plasmids.)

2017 Measurement Kit

Along with the Distribution Plates, teams will receive a Measurement Kit from iGEM HQ in the Distribution Kit. For more details, please read the documentation for our 2017 Measurement Kit. This kit is used to generate the standard curve needed to measure your parts. It can also be used to measure GFP more accurately in your own experiments.

InterLab Protocols and Forms

Teams who participate in the 2017 InterLab Study are required to follow the protocols below and to submit data using the forms and files provided. All participating teams must submit data from a plate reader. In addition to the plate reader, you can also send us data from a flow cytometer (a flow cytometer protocol is also provided).

Please note: the protocols have been created using Google Forms. If you require an alternative or cannot view them, please email us at measurement AT igem DOT org for an alternative file format.

Transform the InterLab Parts

Prior to starting the measurement protocols below, please transform the 8 plasmids (Positive Control, Negative Control, Test Device 1, Test Device 2, Test Device 3, Test Device 4, Test Device 5, and Test Device 6 - locations listed below) from Kit Plate 6 or Kit Plate 7 into E. coli DH5-alpha cells. You need to resuspend each plasmid in 10 µL of distilled water (diH₂O). The liquid will turn red to indicate you have successfully resuspended the plasmid (we dry down the plasmids with cresol red dye).

We recommend using our Transformation Protocol, but any plasmid transformation protocol should work well if you have an alternative.

Kit Plate 6 InterLab Part Locations

Positive Control (BBa_I20270): well 20B
Negative Control (BBa_R0040): well 20D
Test Device 1 (BBa_J364000): well 20F
Test Device 2 (BBa_J364001): well 20H
Test Device 3 (BBa_J364002): well 20J
Test Device 4 (BBa_J364003): well 20L
Test Device 5 (BBa_J364004): well 20N
Test Device 6 (BBa_J364005): well 20P

Kit Plate 7 InterLab Part Locations

Positive Control (BBa_I20270): well 21B
Negative Control (BBa_R0040): well 21D
Test Device 1 (BBa_J364000): well 21F
Test Device 2 (BBa_J364001): well 21H
Test Device 3 (BBa_J364002): well 21J
Test Device 4 (BBa_J364003): well 21L
Test Device 5 (BBa_J364004): well 21N
Test Device 6 (BBa_J364005): well 21P

Antibiotic Resistance: Each device is in the pSB1C3 plasmid backbone, so transform all of the devices onto plates containing chloramphenicol.

If you have any problems with transforming these plasmids, please email Traci (traci AT igem DOT org) for help.

Once your cells are transformed and you have colonies, you can start the protocols below!

Plate Reader

Flow Cytometer

Competition/InterLab Study