Reproducibility of fluorescent expression from engineered biological constructs in E. coli

We present results of the first large-scale interlaboratory study carried out in synthetic biology, as part of the 2014 and 2015 International Genetically Engineered Machine (iGEM) competitions. Participants at 88 institutions around the world measured fluorescence from three engineered constitutive constructs in E. coli. Few participants were able to measure absolute fluorescence, so data was analyzed in terms of ratios. Precision was strongly related to fluorescent strength, ranging from 1.54-fold standard deviation for the ratio between strong promoters to 5.75-fold for the ratio between the strongest and weakest promoter, and while host strain did not affect expression ratios, choice of instrument did. This result shows that high quantitative precision and reproducibility of results is possible, while at the same time indicating areas needing improved laboratory practices

Correction: Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli.

[This corrects the article DOI: 10.1371/journal.pone.0150182.]

Instrument-to-instrument variation within a single team.

<p>Each column represents a set of replicates measured by the same laboratory on different instruments. Blue circles are the ratios for individual instruments, normalized by mean ratio; the red line spans ±1 std.dev.</p

Instruments reported in 2014 and 2015 iGEM Interlab Studies.

<p>Instruments reported in 2014 and 2015 iGEM Interlab Studies.</p

Distributions of data partitioned by <i>E. coli</i> strain for each ratio, omitting outliers.

<p>Note that the BL21 subset in 2014 has only a single non-outlier data point and may thus be effectively ignored.</p

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

<p>Constitutive fluorescence constructs measured in the 2014 and 2015 iGEM Interlab Studies.</p

Log-Normal distribution diagrams for fluorescence expression ratios.

<p>The mostly linear structure indicates that variation in expression does generally conform closely to log-normal distribution, with more vertical clustering indicating tighter distributions. Data points significantly off the line will be considered outliers and not used in further analysis of these distributions.</p

Strains reported in 2014 and 2015 iGEM Interlab Studies.

<p>Strains reported in 2014 and 2015 iGEM Interlab Studies.</p

Relationship between mean ratio and precision.

<p>The lower another promoter’s expression is in relation to the strong construct, the higher the variation in measurement: standard deviation grows approximately in proportion to the square root of the mean ratio.</p

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

<p>Note that some subsets have only one or zero non-outlier data points and may thus be effectively ignored.</p