Name: high throughput screening of fungal endoglucanase activity in escherichia coli
Uploaded: 2011-08-13T15:00:00
Description: Watch this Scientific Journal Video about High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli at JoVE.com

这项工作是由戈登和贝蒂摩尔基金会，由UNCF /默克科学倡议。

1。筛选板的制备<ol><li>新增25G的LB生长介质，15G琼脂培养基，并1.5克羧甲基纤维素（CMC）基板1L蒸馏水，高压灭菌消毒。 </li><li>高压灭菌后，允许中等降温，并添加适当的抗生素，IPTG至终浓度为100μm。 </li><li>倒入200ML中型每到5个大型广场培养皿/生物测定托盘（240 × 240 × 20mm或更大）。允许完全干燥板。 </li></ol> 2。内切葡聚糖酶库的生成和屏幕<ol><li>生成的内切葡聚糖酶催化...

<ol>
	<li>Atsumi, S., Hanai, T., Liao, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-fermentative+pathways+for+synthesis+of+branched-chain+higher+alcohols+as+biofuels.">Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels.</a> Nature. 451, 86-89 (2008).</li><li>Wilson, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellulases+and+biofuels.">Cellulases and biofuels.</a> Curr Opin Biotechnol. 20, 295-299 (2009).</li><li>Qin, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+endoglucanase+II+from+Trichoderma+reesei+to+improve+the+catalytic+efficiency+at+a+higher+pH+optimum.">Engineering endoglucanase II from Trichoderma reesei to improve the catalytic efficiency at a higher pH optimum.</a> J Biotechnol. 135, 190-195 (2008).</li><li>Nakazawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+evolution+of+endoglucanase+III+(Cel12A)+from+Trichoderma+reesei.">Directed evolution of endoglucanase III (Cel12A) from Trichoderma reesei.</a> Appl Microbiol Biotechnol. 83, 649-657 (2009).</li><li>Heinzelman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+family+of+thermostable+fungal+cellulases+created+by+structure-guided+recombination.">A family of thermostable fungal cellulases created by structure-guided recombination.</a> Proc Natl Acad Sci. 106, 5610-5615 (2009).</li><li>Heinzelman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SCHEMA+recombination+of+a+fungal+cellulase+uncovers+a+single+mutation+that+contributes+markedly+to+stability.">SCHEMA recombination of a fungal cellulase uncovers a single mutation that contributes markedly to stability.</a> J Biol Chem. 284, 26229-26233 (2009).</li><li>Lantz, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypocrea+jecorina+CEL6A+protein+engineering.">Hypocrea jecorina CEL6A protein engineering.</a> Biotechnol Biofuels. 8, 3-20 (2010).</li><li>Mahadevan, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-directed+mutagenesis+and+CBM+engineering+of+Cel5A+(Thermotoga+maritima).">Site-directed mutagenesis and CBM engineering of Cel5A (Thermotoga maritima).</a> FEMS Microbiol Lett. 287, 205-211 (2008).</li><li>Nakazawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+catalytic+domains+of+Trichoderma+reesei+endoglucanase+I,+II,+and+III,+expressed+in+Escherichia+coli.">Characterization of the catalytic domains of Trichoderma reesei endoglucanase I, II, and III, expressed in Escherichia coli.</a> Appl Microbiol Biotechnol. 81, 681-689 (2008).</li><li>Wang, H., Jones, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+the+Macrophomina+phaseolina+endoglucanase+(EGL+1)+gene+product+in+bacterial+and+yeast+expression+systems.">Properties of the Macrophomina phaseolina endoglucanase (EGL 1) gene product in bacterial and yeast expression systems.</a> Appl Biochem Biotechnol. 81, 153-160 (1999).</li><li>Howard, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme+activity+of+a+Phanerochaete+chrysosporium+cellobiohydrolase+(CBHI.1)+expressed+as+a+heterologous+protein+from+Escherichia+coli.">Enzyme activity of a Phanerochaete chrysosporium cellobiohydrolase (CBHI.1) expressed as a heterologous protein from Escherichia coli.</a> Afr J Biotechnol. 2, 296-300 (2003).</li><li>Howard, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+a+chimeric+Phanerochaete+chrysosporium+cellobiohydrolase+expressed+from+Escherichia+coli.">Characterisation of a chimeric Phanerochaete chrysosporium cellobiohydrolase expressed from Escherichia coli.</a> Afr J Biotechnol. 3, 349-352 (2004).</li><li>Teather, R. M., Wood, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Congo+red-polysaccharide+interactions+in+enumeration+and+characterization+of+cellulolytic+bacteria+from+the+bovine+rumen.">Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen.</a> Appl Env Microbiol. 43, 777-780 (1982).</li><li>Gilkes, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mode+of+action+and+substrate+specificities+of+cellulases+from+cloned+bacterial+genes.">Mode of action and substrate specificities of cellulases from cloned bacterial genes.</a> J Bio Chem. 259, 10455-10459 (1984).</li></ol>

这里所描述的协议，允许快速和高活性酶的吞吐量鉴定，用最少的操作。活动检测是相当敏感，定性地反映细胞内的活动量。它的易用性使得这种方法适合广泛的酶库，只能通过在大肠杆菌中表达功能的限制大肠杆菌 。此外，这种筛查活动并不限于真菌纤维素酶，但可适应任何内切葡聚糖酶的活性，或任何纤维素酶是一种可溶性底物（如CMC）。

<table border="1"><tbody><tr><th>试剂</th><th>公司</th><th>产品编号</th></tr><tr><td>经IPTG </td><td>西格玛</td><td> I1284 </td></tr><tr><td>刚果红</td><td>西格玛</td><td> C6277 </td></tr><tr><td>羧甲基纤维素</td><td>西格玛</td><td> 360384 </td></tr><tr><td>氯化钠</td><td>西格玛</td><td> S1679 </td></tr><tr><td>细菌用的琼脂</td><td> BD公司</td><td> 214030 </td></tr><tr><td>生物测定板</td><td> Thermo Scientific的</td><td> 240845 </td></tr></tbody></table>

high throughput screening of fungal endoglucanase activity in escherichia coli

纤维素酶（endoglucanases，cellobiohydrolases，和β-葡萄糖苷酶）水解成组成糖的纤维素，这反过来又可以转换成燃料醇 1 。酶解纤维素生物质来提供可再生能源的潜力，加紧努力，以工程师为经济的燃料生产 2纤维素酶。特别感兴趣的是真菌纤维素酶 3-8，目前已经被用于食品和纺织品加工工业。 确定之间的突变纤维素库积极变种，工程过程是至关重要的;积极的突变体，可进一步测试，性能改进和/或遭受额外的突变。真菌纤维素酶的高效的工程已经阻碍了原生生物体的遗传工具的缺乏和难以表达的酶在异源主机。近日，森川和他的同事开发出一种在大肠杆菌中的表达方法H. jecorina 3,9，一个重要的工业真菌的能力，分泌大量的纤维素endoglucanases大肠杆菌的催化结构域。功能性大肠杆菌中的表达，也为其他真菌的纤维素酶， 包括Macrophomina phaseolina 10 和黄孢原 毛平革 11-12报告。 我们目前在大肠杆菌中的真菌内切葡聚糖酶的活性高通量筛选的方法大肠杆菌 。 （ 图1）此方法使用的常见的微生物染料刚果红（CR），以可视化的羧甲基纤维素（CMC）的固体培养基上生长的细胞的酶降解。酶活性测定需要廉价的试剂，最小操纵，退化区，在殖民地网站（“光环”），并给出明确的结果。虽然酶活性的定量测量，不能用这种方法确定的，我们发现，光环的大小总在细胞内的酶的活性相关。个别阳性克隆进一步鉴定，确定，相对蛋白健身。 CMC / CR传统的细菌全细胞活性测定13涉及泼在琼脂含有CMC的殖民地，这是受交叉污染，或在CMC琼脂井，这是不适合进行大规模的试验培育文化。在这里，我们报告的改进协议，修改现有14纤维素酶洗方法：CMC琼脂平板上生长的CR染色前去除。我们的协议大大降低了交叉污染和高度的可扩展性，使成千上万的克隆快速筛选。除了​​到 H jecorina酶 ，我们都表示并筛选的嗜热子囊菌斜卧青霉 （ 如图2所示）内切葡聚糖酶变种，这表明本议定书适用范围从一个生物体的酶。

Watch this Scientific Journal Video about High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli at JoVE.com

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

我们描述了一种低成本，高通量方法真菌内切葡聚糖酶的活性在屏幕<em&gt; E。大肠杆菌。</em&gt;该方法依赖于一个简单的基质降解的视觉读数，不需要酶的纯化，并高度可伸缩的。这使得酶变异体的大型图书馆的快速筛选。

真菌内切葡聚糖酶的活性高通量筛选大肠埃希氏菌</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

Research

JoVE Journal

Biology

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

用于测量的活性测定大肠埃希氏菌诱导赖氨酸Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

科研

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A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

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使用自制双辉光萤光素酶检测的高通量筛选功能

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High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

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高通量定量表达筛选与纯化应用到重组二硫键丰富的蛇毒蛋白产于<em&gt; E。大肠杆菌</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

蛋白质的共表达了多方面的好处<em&gt;大肠杆菌</em

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...

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高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

激酶底物对鉴定使用高通量筛选


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...