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

Kelly JR, Rubin AJ, Davis JH, Ajo-Franklin CM, Cumbers J, Czar MJ, de Mora K, Glieberman AL, Monie DD, Endy D - J Biol Eng (2009)

Bottom Line: We found that the absolute activity of BioBrick promoters varies across experimental conditions and measurement instruments.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 approximately 50%.We distributed a set of test promoters to multiple labs and found good agreement in the reported relative activities of promoters so measured.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA. jrkelly@gmail.com

ABSTRACT

Background: The 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.

Results: We 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 approximately 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.

Conclusion: Relative 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.

No MeSH data available.


Promoter collections can be readily characterized via Relative Promoter Units (RPUs). The five promoters labeled J23### are from a constitutive promoter library and R0040 and R0011 are tet- and lac-repressible promoters, respectively. The activity of the promoters was measured in relative promoter units (RPUs). This collection of promoter may itself be useful for tuning gene expression in engineered systems. The error bars represent the 95% confidence interval of the mean based on nine replicates.
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Figure 3: Promoter collections can be readily characterized via Relative Promoter Units (RPUs). The five promoters labeled J23### are from a constitutive promoter library and R0040 and R0011 are tet- and lac-repressible promoters, respectively. The activity of the promoters was measured in relative promoter units (RPUs). This collection of promoter may itself be useful for tuning gene expression in engineered systems. The error bars represent the 95% confidence interval of the mean based on nine replicates.

Mentions: Given that many laboratories could coordinate their measurement of promoter activities, we sought to prepare tools that would facilitate the widespread adoption of relative promoter activity measurements. To do this we first measured the relative activities of a set of seven representative promoters obtained from the Registry of Standard Biological Parts (Figure 3) [8]. These promoters included members of a constitutive promoter library (BBa_J23100 – BBa_J23119, constructed by JC Anderson) as well as the commonly used Tet repressor (BBa_R0040) and Lac repressor (BBa_R0011) regulated promoters [29]. The regulated promoters were tested in the absence of their cognate repressor proteins. Such libraries of characterized promoters have been shown to be valuable to researchers for tuning biochemical networks to optimize the synthesis of products of interest [20,30]. We measured the relative promoter activities by calculating the steady-state GFP synthesis rates (Methods) and converting these rates to RPUs. Nine independent clones were characterized across three separate experimental runs for each promoter tested. The promoters ranged in activity from 0.026 ± 0.003 to 1.45 ± 0.095 RPUs (uncertainties represent 95% confidence interval of the mean). The GFP expression level from one promoter (BBa_J23113) was statistically equivalent within measurement error to the expression level of the negative control (TOP10).


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

Kelly JR, Rubin AJ, Davis JH, Ajo-Franklin CM, Cumbers J, Czar MJ, de Mora K, Glieberman AL, Monie DD, Endy D - J Biol Eng (2009)

Promoter collections can be readily characterized via Relative Promoter Units (RPUs). The five promoters labeled J23### are from a constitutive promoter library and R0040 and R0011 are tet- and lac-repressible promoters, respectively. The activity of the promoters was measured in relative promoter units (RPUs). This collection of promoter may itself be useful for tuning gene expression in engineered systems. The error bars represent the 95% confidence interval of the mean based on nine replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2683166&req=5

Figure 3: Promoter collections can be readily characterized via Relative Promoter Units (RPUs). The five promoters labeled J23### are from a constitutive promoter library and R0040 and R0011 are tet- and lac-repressible promoters, respectively. The activity of the promoters was measured in relative promoter units (RPUs). This collection of promoter may itself be useful for tuning gene expression in engineered systems. The error bars represent the 95% confidence interval of the mean based on nine replicates.
Mentions: Given that many laboratories could coordinate their measurement of promoter activities, we sought to prepare tools that would facilitate the widespread adoption of relative promoter activity measurements. To do this we first measured the relative activities of a set of seven representative promoters obtained from the Registry of Standard Biological Parts (Figure 3) [8]. These promoters included members of a constitutive promoter library (BBa_J23100 – BBa_J23119, constructed by JC Anderson) as well as the commonly used Tet repressor (BBa_R0040) and Lac repressor (BBa_R0011) regulated promoters [29]. The regulated promoters were tested in the absence of their cognate repressor proteins. Such libraries of characterized promoters have been shown to be valuable to researchers for tuning biochemical networks to optimize the synthesis of products of interest [20,30]. We measured the relative promoter activities by calculating the steady-state GFP synthesis rates (Methods) and converting these rates to RPUs. Nine independent clones were characterized across three separate experimental runs for each promoter tested. The promoters ranged in activity from 0.026 ± 0.003 to 1.45 ± 0.095 RPUs (uncertainties represent 95% confidence interval of the mean). The GFP expression level from one promoter (BBa_J23113) was statistically equivalent within measurement error to the expression level of the negative control (TOP10).

Bottom Line: We found that the absolute activity of BioBrick promoters varies across experimental conditions and measurement instruments.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 approximately 50%.We distributed a set of test promoters to multiple labs and found good agreement in the reported relative activities of promoters so measured.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA. jrkelly@gmail.com

ABSTRACT

Background: The 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.

Results: We 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 approximately 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.

Conclusion: Relative 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.

No MeSH data available.