Rational diversification of a promoter providing fine-tuned expression and orthogonal regulation for synthetic biology.
Rational diversification of a promoter providing fine-tuned expression and orthogonal regulation for synthetic biology.
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DOI：
10.1371/journal.pone.0033279
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Ellis T
中科院分区：
文献类型：
--
作者：
Blount BA;Weenink T;Vasylechko S;Ellis T
酵母是合成生物学开发和应用的理想生物体，但仍然存在相对较少的适合于这种底盘精确工程的良好表征的生物部分。为了解决这一当前的需求，我们在这里提出了一种策略，需要一个单一的生物学部分，启动子，并重新设计它，以产生一个精细分级的输出范围启动子库和新的调节启动子所需的正交合成生物学应用。通过生物信息学方法鉴定了高度组成型酿酒酵母启动子PFY 1 p，对其核心序列进行了体内表征并多样化，创建了一个36个成员的启动子库。TetR调控被引入PFY1p以创建在逆变器装置中起作用的合成诱导型启动子（iPFY1p）。然后，使用可定制的转录激活因子样效应子（TALE）首次证明了合成启动子的正交和可扩展调节，所述可定制的转录激活因子样效应子（TALE）被修饰和设计为充当基于特定PFY1的启动子的正交阻遏物。使启动子在其核心序列多样化，然后独立地将转录激活因子样正交阻遏物（TALOR）靶向几乎任何这些序列的能力显示出对编码复杂的“多线”逻辑功能的未来合成基因网络的设计和构建的巨大希望。
Yeast is an ideal organism for the development and application of synthetic biology, yet there remain relatively few well-characterised biological parts suitable for precise engineering of this chassis. In order to address this current need, we present here a strategy that takes a single biological part, a promoter, and re-engineers it to produce a fine-graded output range promoter library and new regulated promoters desirable for orthogonal synthetic biology applications. A highly constitutive Saccharomyces cerevisiae promoter, PFY1p, was identified by bioinformatic approaches, characterised in vivo and diversified at its core sequence to create a 36-member promoter library. TetR regulation was introduced into PFY1p to create a synthetic inducible promoter (iPFY1p) that functions in an inverter device. Orthogonal and scalable regulation of synthetic promoters was then demonstrated for the first time using customisable Transcription Activator-Like Effectors (TALEs) modified and designed to act as orthogonal repressors for specific PFY1-based promoters. The ability to diversify a promoter at its core sequences and then independently target Transcription Activator-Like Orthogonal Repressors (TALORs) to virtually any of these sequences shows great promise toward the design and construction of future synthetic gene networks that encode complex “multi-wire” logic functions.
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