A roadmap for gene system development in Clostridium.
A roadmap for gene system development in Clostridium.
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DOI：
10.1016/j.anaerobe.2016.05.011
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发表时间：
2016-10
期刊：
影响因子：
2.3
通讯作者：
Zhang Y
中科院分区：
文献类型：
--
作者：
Minton NP;Ehsaan M;Humphreys CM;Little GT;Baker J;Henstra AM;Liew F;Kelly ML;Sheng L;Schwarz K;Zhang Y
梭状芽孢杆菌既是英雄也是恶棍。一些会导致严重的人类和动物疾病，存在于肠道微生物群中的那些通常有助于健康和福祉，而另一些则代表了用于生产化学品和燃料的有用工业底盘。为了理解、对抗或利用，存在对可用于定向或随机基因组修饰的有效系统的基本要求。我们已经制定了一个简单的路线图，从而可以开发和部署必要的基因系统。其核心是使用“假自杀”载体和创建pyrE突变体（尿嘧啶营养缺陷型），最初由ClosTron技术辅助，但最终使用称为ACE（等位基因偶联交换）的特殊形式的等位基因交换。所有的突变体，无论使用何种诱变剂，都是在这种宿主中产生的。这是因为通过使用ACE载体，突变体可以伴随pyrE等位基因的校正和尿嘧啶原养型的恢复而快速补充。这避免了在高拷贝数质粒中经常观察到的表型效应，并且无需添加抗生素以确保质粒保留。一旦可用，pyrE主机就可以用于稳定地插入各种特定于应用程序的模块。实例包括允许部署水手转座子的σ因子、参与生物质解构的水解酶和癌症递送载体中的治疗基因。到目前为止，只要获得DNA转移，我们还没有遇到任何梭菌物种，这种技术不能应用。这些包括艰难梭菌、丙酮丁醇梭菌、拜氏梭菌、肉毒梭菌、产气荚膜梭菌、生孢梭菌、巴氏梭菌、扬氏梭菌、自产乙醇梭菌和甚至热葡糖苷酸土芽孢杆菌。梭菌基因系统开发和应用的简单路线图。使用pyrE等位基因和假自杀载体的等位基因交换。使用等位基因偶联交换（ACE）敲除和敲入。通过基因组插入进行互补研究。应用特定模块的基因组插入。
Clostridium species are both heroes and villains. Some cause serious human and animal diseases, those present in the gut microbiota generally contribute to health and wellbeing, while others represent useful industrial chassis for the production of chemicals and fuels. To understand, counter or exploit, there is a fundamental requirement for effective systems that may be used for directed or random genome modifications. We have formulated a simple roadmap whereby the necessary gene systems maybe developed and deployed. At its heart is the use of ‘pseudo-suicide’ vectors and the creation of a pyrE mutant (a uracil auxotroph), initially aided by ClosTron technology, but ultimately made using a special form of allelic exchange termed ACE (Allele-Coupled Exchange). All mutants, regardless of the mutagen employed, are made in this host. This is because through the use of ACE vectors, mutants can be rapidly complemented concomitant with correction of the pyrE allele and restoration of uracil prototrophy. This avoids the phenotypic effects frequently observed with high copy number plasmids and dispenses with the need to add antibiotic to ensure plasmid retention. Once available, the pyrE host may be used to stably insert all manner of application specific modules. Examples include, a sigma factor to allow deployment of a mariner transposon, hydrolases involved in biomass deconstruction and therapeutic genes in cancer delivery vehicles. To date, provided DNA transfer is obtained, we have not encountered any clostridial species where this technology cannot be applied. These include, Clostridium difficile, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium botulinum, Clostridium perfringens, Clostridium sporogenes, Clostridium pasteurianum, Clostridium ljungdahlii, Clostridium autoethanogenum and even Geobacillus thermoglucosidasius. A simple roadmap for the development and deployment of gene systems in clostridia. Allelic exchange using pyrE alleles and pseudo-suicide vectors. Knock-out and knock-in using allele-coupled exchange (ACE). Complementation studies through genome insertion. Genome insertion of application specific modules.
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影响因子：
6.3
作者：
Ehsaan M;Kuit W;Zhang Y;Cartman ST;Heap JT;Winzer K;Minton NP
通讯作者：
Minton NP
DOI：
10.1007/978-1-61779-065-2_11
发表时间：
2011-01-01
期刊：
CDNA LIBRARIES: METHODS AND APPLICATIONS
影响因子：
--
作者：
Engler, Carola;Marillonnet, Sylvestre
通讯作者：
Marillonnet, Sylvestre
影响因子：
2.8
作者：
Jennert, KCB;Tardif, C;Young, M
通讯作者：
Young, M
影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.
影响因子：
4.7
作者：
de Kok, Stefan;Stanton, Leslie H.;Chandran, Sunil S.
通讯作者：
Chandran, Sunil S.