Measuring the activity of BioBrick promoters using an in vivo reference standard

By J. Kelly, Adam J. Rubin, Joseph H. Davis, C. Ajo-Franklin, J. Cumbers, M. Czar, Aaron L Glieberman, Dileep D. Monie, Drew Endy. Journal of Biological Engineering, 2009

433 citations recorded by the source corpus.

BackgroundThe engineering of many-component, synthetic biological systems is being made easier by the development of collections of reusable, standard biological parts. However, the complexity of biology makes it difficult to predict the extent to which such efforts will succeed. As a first practical example, the Registry of Standard Biological Parts started at MIT now maintains and distributes thousands of BioBrick™ standard biological parts. However, BioBrick parts are only standardized in terms of how individual parts are physically assembled into multi-component systems, and most parts remain uncharacterized. Standardized tools, techniques, and units of measurement are needed to facilitate the characterization and reuse of parts by independent researchers across many laboratories.ResultsWe found that the absolute activity of BioBrick promoters varies across experimental conditions and measurement instruments. We choose one promoter (BBa_J23101) to serve as an in vivo reference standard for promoter activity. We demonstrated that, by measuring the activity of promoters relative to BBa_J23101, we could reduce variation in reported promoter activity due to differences in test conditions and measurement instruments by ~50%. We defined a Relative Promoter Unit (RPU) in order to report promoter characterization data in compatible units and developed a measurement kit so that researchers might more easily adopt RPU as a standard unit for reporting promoter activity. We distributed a set of test promoters to multiple labs and found good agreement in the reported relative activities of promoters so measured. We also characterized the relative activities of a reference collection of BioBrick promoters in order to further support adoption of RPU-based measurement standards.ConclusionRelative activity measurements based on an in vivoreference standard enables improved measurement of promoter activity given variation in measurement conditions and instruments. These improvements are sufficient to begin to support the measurement of promoter activities across many laboratories. Additional in vivo reference standards for other types of biological functions would seem likely to have similar utility, and could thus improve research on the design, production, and reuse of standard biological parts.

Subfields: Biomedical Engineering, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/448e42a04315305a008ebbf9b41f0e2e7aa1d6ab

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Citations in the corpus

Design, construction and characterization of a set of insulated bacterial promoters (2010, methodology): “For most experiments, the corrected synthesis rate was normalized to the average synthesis rate of the proD promoter, resulting in RPUDrelative promoter units (14).”
PromoterPredict: sequence-based modelling of Escherichia coli σ70 promoter strength yields logarithmic dependence between promoter strength and sequence (2018, background): “The measurement of promoter strength could be done in the manner of Kelly et al. (2009) , where the GFP (reporter gene) synthesis rate is measured per unit biomass, and this could be normalized relative to the reference promoter.”
Genetic programs constructed from layered logic gates in single cells (2012, neutral): “S15) 16 .”
The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes (2012, background): “…is the only thus far whose activity can be expressed in absolute units (i.e. Miller units) and, despite its shortcomings (e.g. lengthy manipulations of cells, lack of an internal standard of reference; 66), it is still a very useful asset for studies on promoters in virtually any biological host.”
Genomic Mining of Prokaryotic Repressors for Orthogonal Logic Gates (2013, methodology): “Degenerate oligonucleotides representing the resulting operator motif were designed to insert the operator motif into a strong, constitutive, synthetic Biobricks BBA_J23119 standard promoter 39 .”