Protocols for implementing an Escherichia coli based TX-TL cell-free expression system for synthetic biology.
Protocols for implementing an Escherichia coli based TX-TL cell-free expression system for synthetic biology.
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DOI：
10.3791/50762
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发表时间：
2013-09-16
期刊：
影响因子：
--
通讯作者：
Noireaux V
中科院分区：
文献类型：
--
作者：
Sun ZZ;Hayes CA;Shin J;Caschera F;Murray RM;Noireaux V
理想的无细胞表达系统理论上可以在受控的体外平台中模拟体内细胞环境。1这对于以受控的方式表达蛋白质和遗传电路以及为合成生物学提供原型环境是有用的。2，3为了实现后一个目标，保持内源性大肠杆菌转录的无细胞表达系统-翻译机制能够比基于T7 RNA聚合酶转录的机制更准确地反映体内细胞动力学。我们描述了一个有效的内源性E。基于大肠杆菌的转录-翻译（TX-TL）无细胞表达系统，其可以产生与基于T7的系统等量的蛋白质，与类似的商业系统相比成本降低98%。4，5描述了缓冲液和粗细胞提取物的制备，以及三管TX-TL反应的执行。整个方案需要五天的时间来准备，并在一次制备中产生足够的材料用于多达3000个单一反应。一旦准备好，每个反应从设置到数据收集和分析需要不到8小时。对E.大肠杆菌，如lac/泰特阻遏物和T7 RNA聚合酶。6内源特性，如mRNA和DNA降解速率，也可以调整。7 TX-TL无细胞表达系统已被证明用于大规模电路组装，探索生物现象，以及在T7和内源启动子下表达蛋白质。6，8伴随的数学模型是可用的。9，[10]所得到的系统在合成生物学中作为原型环境或“TX-TL生物分子试验板”具有独特的应用。"
Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."
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影响因子：
5.6
作者：
Shin J;Noireaux V
通讯作者：
Noireaux V
影响因子：
5.6
作者：
Shin J;Noireaux V
通讯作者：
Noireaux V
影响因子：
7.7
作者：
Jewett, Michael C.;Forster, Anthony C.
通讯作者：
Forster, Anthony C.
影响因子：
64.8
作者：
Becskei, A;Serrano, L
通讯作者：
Serrano, L
DOI：
10.1073/pnas.2135496100
发表时间：
2003-10-28
影响因子：
11.1
作者：
Noireaux, V;Bar-Ziv, R;Libchaber, A
通讯作者：
Libchaber, A