一个Biomining从宏基因组库的纤维素酶的活性高通量筛选
Summary
该协议描述了一种在大肠杆菌中表达了宏基因组文库的纤维素活动的高通量筛选。屏幕解决方案的基础和高度自动化，并采用一锅煮化学与最后一个测吸光度读数在384孔板。
Abstract
纤维素，有机碳在这个星球上最丰富的来源，具有广泛的工业应用日益重视生物燃料生产 1 。修改或降解纤维素的化学方法通常需要强酸和高温。因此，酶的方法已经成为突出的生物转化过程中。虽然已经有点有效识别来自细菌和真菌菌株的积极纤维素酶，微生物在自然界中的绝大多数抵制实验室培养。环境基因组学，又称宏基因组，筛选方法有很大的希望，在弥合在培育新型生物转化酶的搜索差距。宏基因组筛选方法已成功地从环境恢复新颖纤维素酶作为土壤2，水牛瘤胃3和白蚁后肠4使用羧甲基纤维素钠（CMC）的琼脂板用刚果红染料（5 Teather和木材的方法的基础上）染色不同。然而，中央军委的方法是在吞吐量有限，不定量，并表现出低信号信噪比6。其他方法已经7,8报道，但每次使用的琼脂平板为基础的检测，这是为大型插入基因组文库的高通量筛选的不良。在这里，我们提出了一个解决方案，基于纤维素酶的活动，使用显色二硝基苯酚（DNP）cellobioside基板9屏幕。我们的资料库，克隆到fosmid增加检测的灵敏度，通过拷贝数诱导 10 pCC1复制控制。该方法使用一锅煮化学作为测吸光度提供的最终读数384孔板。定量，灵敏，吞吐量高达每天100X 384孔板使用液体处理和附有堆垛系统酶标仪自动读数。
Protocol
本议定书开始之前，您将需要您的宏基因组文库在384孔板格式存储。在我们的研究中，我们结合使用噬菌体T1耐TransforMax EPI300 – T1 ř 大肠杆菌 pCC1复制fosmid矢量控制大肠杆菌细胞库的主机和存储我们的板在-80℃11。 1。复制的宏基因组文库板除霜板包含您的图书馆在37℃，约20分钟，或者直到所有的水井都解冻。 消毒15分钟的机器人上…
Discussion
在大肠杆菌中表达了一个高通量筛选，从一个大的插入基因组DNA的宏基因组文库的快速检测纤维素活动大肠杆菌是在本议定书。这个方法是在CMC /刚果红在文献中常用的检测改善。这是解决方案为基础，并允许一锅煮化学筛选与定量分析，使读者从板块的吸光度读数最终输出384孔板。在这个过程的每一步自动化允许超过25每小时384板无监督检查。从软件中的数据可以很容易地导出到Mic…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者要感谢史蒂夫威瑟斯博士和香港明陈提供的DNP – Cellobioside基板。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| qPix2 | Genetix | With 384-pin gridding head | ||
| qFill3 | Genetix | With 384-well manifold | ||
| Varioskan | Thermo-Fisher | |||
| RapidStak | Thermo-Fisher | Connected to Varioskan | ||
| Micro90 Detergent | Cole-Parmer | 18100-00 | Diluted to 2% in water | |
| Ethanol | Major Lab Supplier | Diluted to 80% in water | ||
| Chloramphenicol | Sigma | C0378 | 12.5mg/mL in ethanol | |
| LB broth, Miller | Fisher | BP1426-2 | 25g/L, autoclaved | |
| 384-well flat bottom plates | Corning | 3680 | ||
| L-(+)-Arabinose | Sigma | A3256 | 100mg/mL in water | |
| Potassium Acetate | Fisher | P171 | 50mM in water, autoclaved, adjusted to pH 5.5 with HCl | |
| Triton X-100 | Fisher | BP151 | ||
| Trizma hydrochloride | Sigma | T3253 | In TE buffer solution, 100mM | |
| EDTA disodium salt | Sigma | E5134 | In TE buffer solution, 10mM | |
| 2,4-dinitrophenyl cellobioside | Provided by Dr. Steve Withers, UBC | |||
| Dimethyl Sulfoxide | Sigma | D8418 |
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Tags
High Throughput ScreenBiomining Cellulase ActivityMetagenomic LibrariesCelluloseIndustrial ApplicationsBiofuel ProductionChemical MethodsEnzymatic MethodsCellulasesBacterial IsolatesFungal IsolatesLaboratory CultivationEnvironmental Genomic ScreeningMetagenomic Screening ApproachesSoilsBuffalo RumenTermite Hind-gutCarboxymethylcellulose (CMC) Agar PlatesCongo Red DyeMethod Of Teather And WoodSignal To Noise RatioChromogenic Dinitrophenol (DNP)-cellobioside SubstratePCC1 C
Cite This Article
Mewis, K., Taupp, M., Hallam, S. J. A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries. J. Vis. Exp. (48), e2461, doi:10.3791/2461 (2011).