Characterization of 582 natural and synthetic terminators and quantification of their design constraints
Characterization of 582 natural and synthetic terminators and quantification of their design constraints
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DOI：
10.1038/nmeth.2515
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发表时间：
2013-07-01
期刊：
影响因子：
48
通讯作者：
Voigt, Christopher A.
中科院分区：
文献类型：
--
作者：
Chen, Ying-Ja;Liu, Peng;Voigt, Christopher A.
大型基因工程项目需要更多的顺反子，因此需要更强大和可靠的转录终止子。我们已经测量了终止子库的强度，包括227个在大肠杆菌中注释的终止子-其中90个我们还测试了反向终止子和265个合成终止子。在该文库中，我们发现了39个强终止子，其在下游表达中产生>50倍的减少，当在设计中一起使用时，其具有足够的序列多样性以减少同源重组。我们利用这些数据来确定终止子序列如何有助于其强度。将主要参数纳入生物物理模型，该模型考虑发夹在U形束从DNA的位移中的作用。不同强度的许多终止子的可用性，以及对它们的性质的序列依赖性的理解，将扩展它们在合成顺反子的正向设计中的可用性。
Large genetic engineering projects require more cistrons and consequently more strong and reliable transcriptional terminators. We have measured the strengths of a library of terminators, including 227 that are annotated in Escherichia coli-90 of which we also tested in the reverse orientation-and 265 synthetic terminators. Within this library we found 39 strong terminators, yielding >50-fold reduction in downstream expression, that have sufficient sequence diversity to reduce homologous recombination when used together in a design. We used these data to determine how the terminator sequence contributes to its strength. The dominant parameters were incorporated into a biophysical model that considers the role of the hairpin in the displacement of the U-tract from the DNA. The availability of many terminators of varying strength, as well as an understanding of the sequence dependence of their properties, will extend their usability in the forward design of synthetic cistrons.