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Student teams take on synbio reproducibility problem

Synthetic biology aims to engineer biological cells to perform many different useful functions, but before they can be used commercially or clinically they are extensively tested in the lab. Fluorescent proteins serve as our main tool for monitoring the output of the engineered circuits we put in cell. Most data you see on a synthetic biology circuit is produced by measuring fluorescence from proteins like green fluorescent protein (GFP).

But what if we are not all measuring the fluorescence in the same way with the same equipment? Is the data comparable across labs and users?

Well, over the last two years iGEM teams around the world have been working to find out just how reproducible fluorescent proteins measurements are. They distributed testing plasmids and compared results across labs, measurement instruments, genetic parts, and E. coli strains.  It’s a thorough 2 year study of interlab variability, and the results are out in PLOS ONE, “Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli“. 


Reproduced Fig 1. Constitutive fluorescence constructs measured in the 2014 and 2015 iGEM Interlab Studies.

The importance of fluorescent proteins in synthetic biology is that they can be programmed to be the output at different parts of a genetic circuit to monitor whether that part is on and how much strongly it is expressing protein. Each fluorescent protein is detected by flashing a laser input that provides energy to the protein that is then released at a specific color. By using different color fluorescent proteins a researcher can monitor the expression of multiple aspects of a circuit. Since this is the basis of most of our data on synthetic biology circuits we need to know how to interpret it. 

If we’re trying to compare data among research labs, we need to have some standardized way to measure and analyze data. This paper is an important step in our understanding of how we make measurements of our synbio circuits.

Reproduced Fig 6. Distributions of data partitioned by measuring instrument for each ratio, omitting outliers.

Instead of comparing the absolute numbers that come off of the machine, they analyzed the ratios of measurements of different promoter strengths. The team showed that different E. coli strains give different fluorescent signals and that different instruments give different results. There are many known differences among strains, but I think fewer people had given thought to the measurement hardware as an issue for reproducibility.

Another interesting point is that variances among different researchers’ measurements  is higher for for the high ratio measurements. This happens because the a large ratios is achieved by comparing a strong and weak promoter, and the measurement for the weak promoter is more noisy.

Reproduced Fig 4. Relationship between mean ratio and precision.
Reproduced Fig 4. Relationship between mean ratio and precision.

The authors recommend three methods to help improve reproducibility in light of their work:

1.Dissemination, training, and standardization around improved protocols for calibration of assays.

2.Removing “craft” from protocol execution.

3.Increased automation of protocols.

It’s great to see iGEM teams collaborate with full time researchers and be able to publish their work. If you’re involved with an iGEM team don’t forget that PLOS is taking submissions until Jan. 28 2017 so you can get your work out there like this interlab reproducibility work.

For more details of this interlab study on fluorescent protein measurements, be sure to read the full article.



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