程序的微生物实验室自适应演化的恒化
Summary
在这里，我们提出了一个协议，以获得使用恒化培养条件下微生物实验室适应性进化。此外，演进的株的基因组分析进行了讨论。
Abstract
Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) refers to the experimental situation in which evolution is observed using living organisms under controlled conditions and stressors; organisms are thereby artificially forced to make evolutionary changes. Microorganisms are subject to a variety of stressors in the environment and are capable of regulating certain stress-inducible proteins to increase their chances of survival. Naturally occurring spontaneous mutations bring about changes in a microorganism’s genome that affect its chances of survival. Long-term exposure to chemostat culture provokes an accumulation of spontaneous mutations and renders the most adaptable strain dominant. Compared to the colony transfer and serial transfer methods, chemostat culture entails the highest number of cell divisions and, therefore, the highest number of diverse populations. Although chemostat culture for ALE requires more complicated culture devices, it is less labor intensive once the operation begins. Comparative genomic and transcriptome analyses of the adapted strain provide evolutionary clues as to how the stressors contribute to mutations that overcome the stress. The goal of the current paper is to bring about accelerated evolution of microorganisms under controlled laboratory conditions.
Introduction
微生物可以生存和适应不同的环境。在严峻的压力，可以通过随机的基因组突变和随后的正选择1-3取得有益的表型发生调整。因此，微生物细胞可以通过改变代谢或调控网络对于最佳生长，这被称为“自适应进化”适应。最近的重要微生物的倾向，比如超级细菌爆发和强大的微生物菌种的发生，关系非常密切的压力条件下的适应性进化。下定义的实验室条件下，我们能够研究分子进化的机制和甚至控制各种应用微生物进化的方向。与多细胞生物，单细胞生物是非常适合自适应进化实验室（ALE），原因如下：他们很快再生，他们认为大量的人口，而且很容易创建和维护坎ogeneous环境。随着DNA测序技术和高通量技术最新进展相结合，ALE允许对基因组的变化，导致系统性监管政策的变化直接观察。突变动力学和人口的多样性也观察到。遗传工程策略可以从ALE菌株4,5的分析来确定。
恒化器培养是用于获得稳态细胞和发酵过程6提高生产率的方法。新鲜培养基，并将该过程（后者包括介质和生物量）中培养液收获。长期培养恒然而，改变了文化的稳态生产力和文化（ 图1a）期间带来的自发突变和选择的积累。在各种选择压力（压力），突变的积累被提高。应力的长期逐渐增加恒化器提供了用于对给定的压力工作的突变，如温度，pH，渗透压，营养饥饿，氧化，有毒最终产品等菌落转移从液体介质中的固体培养基和串行传输（重复的连续选择分批培养），也让研究人员获得进化微生物（ 图1b和1c）。虽然恒化培养需要复杂的方法中，分集的池（复制和人口规模的数目）比由菌落转移和串行传输技术获得更高。稳定的压力接触到单个细胞和培养恒（稳态）进行比较，以一批以文化为基础的技术ALE其他福利过程中的细胞状态下降的变化。经受高琥珀酸条件大肠杆菌应激诱导ALE这篇文章中介绍。
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图1：自适应进化实验室方法 （A）恒化。 （B）串行传送; （C）菌落转移。顶部图说明为ALE方法的概念，底部数字说明ALE期间增长了细胞的数量。 请点击此处查看该图的放大版本。
Protocol
Representative Results
Discussion
微生物能够适应，因为他们的速度快速增长和遗传多样性几乎所有的环境。自适应实验室进化使微生物设计条件，这提供了选择个体生物窝藏自发突变是在给定的条件下有利的方式下发展。
恒化器的技术是用于实现比转移技术人工驱动进化原因如下更健壮：（a）一种稳定的环境 – 因为传输技术基于分批培养物无论是在固体或在液体介质中，该细胞的环境批次期间变化文化而?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This study was financially supported by the Korean Ministry of Science, ICT and Future Planning (Intelligent Synthetic Biology Center program 2012M3A6A8054887). P. Kim was supported by a fellowship from the Catholic University of Korea (2015).
Materials
| Mini-chemostat fermentor | Biotron Inc. | – | manufactured by special order |
| silicon tubing | Cole-Parmer | Masterflex L/S 13 | tubing size can be varied depending on the dilution rate and the size of fermentor jar. |
| reservoir jar | Bellco | Media storage bottle | 20 L |
| chemicals | Sigma-Aldrich | – | reagent grade |
| glucose | Sigma-Aldrich | G5767 | ACS reagent |
| NH4Cl | Sigma-Aldrich | A9434 | for molecular biology, suitable for cell culture, ≥99.5% |
| NaCl | Sigma-Aldrich | 746398 | ACS reagent, ≥99% |
| Na2HPO4·2H2O | Sigma-Aldrich | 4272 | 98.5-101% |
| KH2PO4 | Sigma-Aldrich | 795488 | ACS reagent, ≥99% |
| MgSO4·7H2O | Sigma-Aldrich | 230391 | ACS reagent, ≥98% |
| CaCl2 | Sigma-Aldrich | 793639 | ACS reagent, ≥96% |
| thiamine·HCl | Sigma-Aldrich | T4625 | reagent grade, ≥99% |
| Na2·succinate·6H2O | Sigma-Aldrich | S2378 | ReagentPlus, ≥99% |
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