Measuring the activity of BioBrick promoters using an in vivo reference standard.
Measuring the activity of BioBrick promoters using an in vivo reference standard.
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DOI：
10.1186/1754-1611-3-4
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发表时间：
2009-03-20
影响因子：
5.6
通讯作者：
Endy D
中科院分区：
文献类型：
--
作者：
Kelly JR;Rubin AJ;Davis JH;Ajo-Franklin CM;Cumbers J;Czar MJ;de Mora K;Glieberman AL;Monie DD;Endy D
通过开发可重复使用的标准生物部件集合，多组件合成生物系统的工程变得更加容易。然而，生物学的复杂性使得很难预测这些努力的成功程度。作为第一个实际例子，麻省理工学院启动的标准生物部件注册现在维护和分发数千个 BioBrick™ 标准生物部件。然而，BioBrick 部件仅在单个部件如何物理组装成多组件系统方面进行了标准化，并且大多数部件仍然没有特征。需要标准化的工具、技术和测量单位来促进许多实验室的独立研究人员对零件进行表征和重复使用。我们发现 BioBrick 启动子的绝对活性因实验条件和测量仪器而异。我们选择一种启动子（BBa_J23101）作为启动子活性的体内参考标准。我们证明，通过测量相对于 BBa_J23101 的启动子活性，我们可以将由于测试条件和测量仪器的差异而导致的报告启动子活性的变化减少约 50%。我们定义了相对启动子单位 (RPU)，以便以兼容的单位报告启动子表征数据，并开发了测量套件，以便研究人员可以更轻松地采用 RPU 作为报告启动子活性的标准单位。我们将一组测试启动子分发给多个实验室，并发现如此测量的启动子的报告相对活性具有良好的一致性。我们还描述了 BioBrick 启动子参考集合的相对活性，以进一步支持采用基于 RPU 的测量标准。鉴于测量条件和仪器的变化，基于体内参考标准的相对活性测量能够改进启动子活性的测量。这些改进足以开始支持许多实验室的启动子活性测量。其他类型生物功能的体内参考标准似乎可能具有类似的效用，因此可以改进标准生物部件的设计、生产和再利用的研究。
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. 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 ~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. 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.