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Figure 1:
Reference standards reduce variation in reported promoter activities under different measurement conditions. We measured the activity of 2 promoters, J23101 (white columns) and J23150 (grey columns) under seven different measurement conditions and measurement instruments. We varied the media, temperature, cell strain, and plasmid copy number of the promoter test construct. (A) To estimate the per cell GFP synthesis rate we reported the change in fluorescence over a 1 hour period in exponential phase divided by the average absorbance during this period. The coefficient of variation of the GFP synthesis rates across the seven measurement approaches was 49% for J23101 and 39% for J23150. (B) We used the same data and divided the GFP synthesis rate of J23150 (grey bars) by that of J23101 (white bars) in order to calculate the relative promoter activity of J23150 in RPUs. The coefficient of variation of the relative promoter activity across the seven measurement approaches was only 17% suggesting that the relative promoter activity is less sensitive to conditions then absolute activity measured by per cell GFP synthesis rate.
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Abstract
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.