Composability of regulatory sequences controlling transcription and translation in Escherichia coli
Composability of regulatory sequences controlling transcription and translation in Escherichia coli
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DOI：
10.1073/pnas.1301301110
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发表时间：
2013-08-20
影响因子：
11.1
通讯作者：
Church, George M.
中科院分区：
文献类型：
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作者：
Kosuri, Sriram;Goodman, Daniel B.;Church, George M.
无法准确预测异源基因表达水平阻碍了我们设计生物系统的能力。只有当这些元素组合在一起时表现出可预测的行为时，使用具有良好特征的调节元素才能提供潜在的解决方案。我们合成了12563个共同启动子和核糖体结合位点的组合，并同时测量了整个文库中的DNA、RNA和蛋白质水平。使用一个简单的模型，我们发现RNA和蛋白质的表达分别在80%和64%的时间内达到预期水平的两倍。庞大的数据集允许对全局效应进行量化，例如翻译速率对mRNA稳定性的影响以及mRNA二级结构对翻译速率的影响。然而，最糟糕的5%的构造与预测平均偏离了13倍，这可能会阻碍大规模的基因工程项目。这种方法的易用性和可扩展性表明，我们可以筛选合成库，以获得所需的行为，而不是依赖于预测或标准化。
The inability to predict heterologous gene expression levels precisely hinders our ability to engineer biological systems. Using well-characterized regulatory elements offers a potential solution only if such elements behave predictably when combined. We synthesized 12,563 combinations of common promoters and ribosome binding sites and simultaneously measured DNA, RNA, and protein levels from the entire library. Using a simple model, we found that RNA and protein expression were within twofold of expected levels 80% and 64% of the time, respectively. The large dataset allowed quantitation of global effects, such as translation rate on mRNA stability and mRNA secondary structure on translation rate. However, the worst 5% of constructs deviated from prediction by 13-fold on average, which could hinder large-scale genetic engineering projects. The ease and scale this of approach indicates that rather than relying on prediction or standardization, we can screen synthetic libraries for desired behavior.