Name: rapid deletion production in fungi via agrobacterium mediated transformation of oscar deletion constructs
Uploaded: 2017-06-12T13:00:00.000+00:00
Duration: 11 min 24 s
Description: Watch this Scientific Journal Video about Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs at JoVE.com

作者感谢以下本科生和高中生在镰刀菌突变体中产生OSCAR突变体:Anjellica Miller，Athar Naseer，Xiu Lin，Katelyn Woodburry，Chelsea Patterson，Kathleen Robertson，Krystina Bradley，Ashton Rogers，Alexis McKensie，Manny Hernandez ，Ashli​​ Crepsac，Jeff Delong，Christian King，Je Jeong，Maria Belding，Christy Burre，Daniel O&#39;Meara，Lauren（Victoria）Cook，Jake Goodman，Sampriti De，Oge Okoye，Alyssa Beckstead，Garrett Hibbs，Nick Goldstein，Caroline Twum ，Chris Benson，Louis Stokes，Hannah Itell，Jane Hulse，Jasim Mohammed，James Loggins，Kelli Russell，Gre&#39;Nisha Jones，Kristin Sheaffer，Mariam Hammady，Ava Wilson，Katrina Bazemore，Toney Harper，Karlin McGhee，Mohmed Momin，Rima Momin ，Thi Ngoc Le和Angel Pham。

1.基因片段的PCR扩增引物设计<ol><li>下载到文字处理文件中，包含开放阅读框（ORF）的目标基因（GOI）的基因组区域和来自真核生物数据库或其他基因组数据资源的每一侧的基因侧翼至少2 kb。 </li><li>突出显示要删除的ORF，并标记起始和终止密码。 </li><li>在下载的序列中识别并突出显示相邻ORF。 </li><li> 使用GOI ORF的2 kb 5&#39;端和引物设计工具（参见材料列表）设计PCR引物对O1和O2，以产生1 kb的最小大小的产物。小心不要影响相邻的ORF。 <ol><li>将2 kb 5&#39;侧面粘贴到"序列条目"窗口中。单击"显示自定义参数"框。输入以下定制设计参数:底漆TM 58（最小），60（opt）和62（最大）;底漆GC 40（min），50（opt）和60（max）;底漆尺寸22（min），24（opt）和26（最大）;放大镜尺寸1250（min），1500（opt）和2000（最大）。 </li><li>选择反向引物（O2）最接近ORF的结果。捕获测定的屏幕截图，并将引物序列复制并粘贴到文字处理文档中。 </li><li>重复3&#39;侧翼的过程以产生引物O3和O4，此时选择将最接近目标ORF的正向引物（O3）放置的结果。 </li></ol></li><li>在引物1至4的5&#39;末端添加适当的连接位点（ 参见表1 ）。 </li><li>还可以在下面的第4部分中设计和订购用于删除确认的ORF特异性引物。参数应与步骤1.4.1中的相同，但必要时使用Amplicon Size 500（min），750（opt）和1000（max）调整ORF的长度。最后，为了确认同源重组体，产生位于染色体外侧的外侧的5&#39;端和3&#39;端引物。这些"出"引物将与之配合使用hygR（210）和hygF（850）（ 图2 ）。 </li><li>订购底漆。 </li></ol> 2.生产OSCAR构造<ol><li>使用高保真Taq，在一个反应​​中使用引物（100μM）O1和O2，在第二次引物O3和O4中进行两个反应，每个用于5&#39;和3&#39;侧翼。反应混合物含有:29.5μL无菌蒸馏水（SDW），5μL10x LA PCR缓冲液，8μL2.5mM dNTP混合物，5μL25mM MgCl 2，1μL模板（50ng /μL），0.5μLhi-保真度Taq ，0.5μLPrimer O1和0.5μL5&#39;侧翼的引物O2（或0.5μL引物O3和0.5μL引物O4用于3&#39;侧翼）使用反应条件如下:94℃1分钟，然后30个循环94℃30秒，60℃30秒，72℃2分钟，然后最终步骤72℃5分钟，然后无限期地保持在10℃。 </li><li>运行0.8％琼脂糖1×TAE（40mM Tris乙酸盐pH8.3,1mM EDTA）凝胶电泳，在标准微型凝胶装置中约125Vh，以测定产物的大小和相对浓度。根据制造商的说明书，用非乙腈染色将凝胶染色。在紫外线照明器上可视化。 </li><li>如果5&#39;和3&#39;侧面产物大致均匀浓度，将它们组合并使用亲和柱试剂盒（或PEG沉淀90μL组合PCR产物，240μLTE缓冲液pH7.5,160μL30％聚乙二醇PEG8000 30mM MgCl 2 ）根据制造商的方案共同纯化PCR产物。如果它们不是相同的浓度，则所有的低浓度产物与来自较高产量反应的估计等量的产物结合。 </li><li>使用分光光度计测量纯化的DNA浓度。 </li><li>通过混合以下物质进行BP反应:将1μL的60ng /μLpOSCAR，2μL的60ng /μLpA-Hyg-OSCAR，1μL60 ng /μL组合侧腹，1μL克隆酶。在25°C孵育16 h。通过加入0.5μL蛋白酶K终止反应，并在37℃下孵育10分钟。 </li><li>改变高素质大肠杆菌细胞成功地使用市售的感受态细胞和自制的DH5αCaCl 2感受态细胞作为接受者，如制造商的说明书所述。在含有100μg/ mL壮观霉素（Spec）的低钠（0.5g / L NaCl）LB上。 </li><li>通过执行上面产生的菌落的DNA微量排泄10来鉴定正确的构建体。用Hin dIII和Kpn I进行双重消化 ，如下:移液5μLDNA，2 U酶，2μL适当的10倍缓冲液，SDW至20μL总体积。这从载体（ 约 7kb）释放插入片段（约1.5kb的1.5kb基因侧翼）并切割其中的任何位点侧面可以提供可预测的带尺寸。 </li><li>序列11选择显示适当条带图案的克隆。 </li></ol> 3. 农杆菌介导的真菌转化（ATMT） <ol><li>用OSCAR缺失构建体转化根癌土壤杆菌菌株AGL1。 </li><li> 用含有GOI OSCAR缺失质粒的AGL1转化真菌。要执行以下步骤: <ol><li>用2×10 6孢子/ mL的无菌水制备真菌孢子悬浮液。在血细胞计数器或自动细胞计数器上量化孢子。将其放入1.5 mL微量离心管中。这个设置是4个转换板。 </li><li>使用蓝色的无菌一次性回路，从2-3天龄的LB-Spec 100培养基中添加容易看到的含有AGL1的缺失质粒（应覆盖环直径约20％）的糖（见Maateials列表的组成）含有板并混合到孢子悬浮液中。漩涡直到细菌细胞分散（ 约 5分钟中等速度）。 </li><li>将100μL的分生孢子 - Agro悬浮液移至放置在含有共培养培养基12的四个6cm培养皿中的每一个上的纤维素膜过滤器的中心。 </li><li>用无菌玻璃珠（约4）涂覆悬浮液以覆盖膜过滤器的整个表面。或者使用传统的吊具工具分散悬架。将膜过滤器在罩中干燥约10分钟，用石蜡包裹。重复步骤3.2.2和3.2.3设置多个转换。 </li><li>在室温下孵育板2天，倒置。 </li><li>将膜过滤器转移到含有潮霉素B（Hyg）150μg/ mL，200mM头孢噻肟和100μg/ mL的6cm Aspergillus选择培养基拉氧头孢。在室温下孵育5-7天，然后分离Hyg抗性菌落。 </li><li>用无菌牙签将推定的转化子转移到含有150μg/ mL Hyg，100μg/ mL卡那霉素的6cm土豆葡萄糖琼脂（PDA）板上。 </li></ol></li></ol> 4.通过PCR删除突变体鉴定<ol><li> 通过热解从转化体中提取DNA进行PCR。 <ol><li>一旦转化体在步骤3.2.7的PDA上形成菌落，使用无菌牙签将少量（2×2mm）的菌丝体或酵母细胞从菌落转移到100μL裂解溶液（50mM磷酸钠，pH 7.4,1mM EDTA，5％甘油）中。 </li><li>在85-90℃下孵育混合物20-30分钟。将含有基因组DNA的粗提取物储存在-20℃直到在步骤4.2中使用。 </li></ol></li><li>对每个转化体进行4次PCR反应，每次转染d分别检测5&#39;和3&#39;侧翼的同源重组，一个用于可选择标记（在这种情况下为潮霉素磷酸转移酶），一个用于GOI ORF。 注意:我们通常使用廉价的低保真Taq进行这些反应。反应条件如上述步骤2.1所述，含有SDW20μL，10×Taq缓冲液2.5μL，dNTPS（10mM）0.5μL，自制Taq14,0.5μL，引物A（100μM）0.5μL，引物B（100μM） ）0.5μL，模板0.5μL。较低浓度的引物（10μM）可以很好地使用。 </li><li>运行PCR产物的琼脂糖凝胶（ 例如 0.8％）。删除突变体将产生Hyg带，但不产生ORF产物。异位整合将显示两条带。野生型只会产生ORF带。 </li><li>永久存储删除突变体（和异位转化子或两个用于控件）以备将来使用。 注意:使用15％甘油长期在-80°C下撕裂我们的菌株。 </li></ol>

<ol>
	<li>Paz, Z., Garc&#237;a-Pedrajas, M. D., Andrews, D. L., Klosterman, S. J., Baeza-Monta&#241;ez, L., Gold, 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=One+step+construction+of+Agrobacterium-Recombination-ready-plasmids+(OSCAR),+an+efficient+and+robust+tool+for+ATMT+based+gene+deletion+construction+in+fungi.">One step construction of Agrobacterium-Recombination-ready-plasmids (OSCAR), an efficient and robust tool for ATMT based gene deletion construction in fungi.</a> Fungal Genet Biol. 48 (7), 677-684 (2011).</li><li>Gibson, D. G., Young, L., Chuang, R. Y., Venter, J. C., Hutchison, C. A., Smith, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases.">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nat Methods. 6 (5), 343-345 (2009).</li><li>Klosterman, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+genomics+yields+insights+into+niche+adaptation+of+plant+vascular+wilt+pathogens.">Comparative genomics yields insights into niche adaptation of plant vascular wilt pathogens.</a> PLoS Pathog. 7 (7), (2011).</li><li>Xue, C., Wu, D., Condon, B. J., Bi, Q., Wang, W., Turgeon, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+knockout+in+the+maize+pathogen+Setosphaeria+turcica+using+Agrobacterium+tumefaciens-mediated+transformation.">Efficient gene knockout in the maize pathogen Setosphaeria turcica using Agrobacterium tumefaciens-mediated transformation.</a> Phytopathology. 103 (6), 641-647 (2013).</li><li>Xu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+gene+disruption+methodology+for+the+entomopathogenic+fungus+Metarhizium+robertsii.">A high-throughput gene disruption methodology for the entomopathogenic fungus Metarhizium robertsii.</a> PloS One. 9 (9), (2014).</li><li>Crutcher, F. K., Liu, J., Puckhaber, L. S., Stipanovic, R. D., Bell, A. A., Nichols, 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=FUBT,+a+putative+MFS+transporter,+promotes+secretion+of+fusaric+acid+in+the+cotton+pathogen+Fusarium+oxysporum+f.+sp.+vasinfectum.">FUBT, a putative MFS transporter, promotes secretion of fusaric acid in the cotton pathogen Fusarium oxysporum f. sp. vasinfectum.</a> Microbiology. 161, 875-883 (2015).</li><li>Yu, X., Wang, Y., Pan, J., Wei, D., Zhu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+frequency+of+homologous+gene+disruption+by+single-stranded+DNA+in+the+taxol-producing+fungus+Pestalotiopsis+microspora.">High frequency of homologous gene disruption by single-stranded DNA in the taxol-producing fungus Pestalotiopsis microspora.</a> Ann Microbiol. 65 (4), 2151-2160 (2015).</li><li>Korn, M., Schmidpeter, J., Dahl, M., M&#252;ller, S., Voll, L. M., Koch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Genetic+Screen+for+Pathogenicity+Genes+in+the+Hemibiotrophic+Fungus+Colletotrichum+higginsianum+Identifies+the+Plasma+Membrane+Proton+Pump+Pma2+Required+for+Host+Penetration.">A Genetic Screen for Pathogenicity Genes in the Hemibiotrophic Fungus Colletotrichum higginsianum Identifies the Plasma Membrane Proton Pump Pma2 Required for Host Penetration.</a> PloS One. 10 (5), e0125960 (2015).</li><li>Chettri, 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> Regulation of dothistromin toxin biosynthesis by the pine needle pathogen Dothistroma septosporum: a thesis presented in the partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics at Massey University, Manawatu, New Zealand . , (2014).</li><li>Chen Zhou, ., Yujun Yang, ., Jong, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini-prep+in+ten+minutes.">Mini-prep in ten minutes.</a> Biotechniques. 8 (2), 172 (1990).</li><li>Sanger, F., Nicklen, S., Coulson, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+sequencing+with+chain-terminating+inhibitors.">DNA sequencing with chain-terminating inhibitors.</a> P Natl Acad SciUSA. 74 (12), 5463-5467 (1977).</li><li>Khang, C. H., Park, S. Y., Rho, H. S., Lee, Y. H., Kang, S., Wang, K. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filamentous+fungi+(Magnaporthe+grisea+and+Fusarium+oxysporum).">Filamentous fungi (Magnaporthe grisea and Fusarium oxysporum).</a> Agrobacterium Protocols. 2, 403-420 (2007).</li><li>Zhang, Y. J., Zhang, S., Liu, X. Z., Wang Wen, H. A., M, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+genomic+DNA+extraction+suitable+for+analysis+of+bulk+fungal+strains.">A simple method of genomic DNA extraction suitable for analysis of bulk fungal strains.</a> Lett Appl Microbiol. 51 (1), 114-118 (2010).</li><li>Pluthero, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+purification+of+high-activity+Taq+DNA+polymerase.">Rapid purification of high-activity Taq DNA polymerase.</a> Nucleic Acids Res. 21 (20), 4850-4851 (1993).</li><li>McCluskey, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boosting+Research+and+Industry+by+Providing+Extensive+Resources+for+Fungal+Research.">Boosting Research and Industry by Providing Extensive Resources for Fungal Research.</a> Gene Expression Systems in Fungi: Advancements and Applications. , 361-384 (2016).</li></ol>

作者没有什么可以披露的。

已经成功地应用了农杆菌的一步构建 - 组合就绪质粒（OSCAR），其中越来越多的子囊菌真菌。假设农杆菌介导的转化和同源重组是可能的，该方法还应该容易地应用于担子菌门和其他真菌门（具有适当的启动子驱动选择性标记基因）的物种。已经产生了额外的标记载体以使抗真菌化合物的选择多样化，并且允许产生双重和高级突变体。这些包括抗G418，奈瑟菌素，最近除草剂草铵膦和?...

遗传解剖是确定个体或基因组合的功能重要性的有力方法。了解特定基因作用的标准方法是在任何其他基因中不改变单基因突变体的产生。最强大和最不可能的混杂方法是完全和精确地删除感兴趣的基因开放阅读框架（GOI ORF）而不损害任何其他基因功能。 因为用于缺失质粒生成的标准连接方法需要多个步骤，OSCAR 1的合理性就是产生更快速的体外方法。 图1描述了OSCAR方法中的组装过程。本文描述的方法具有将单个多部分反应中的单个基因缺失载体的快速构建与随后的根癌土壤杆菌介导转录（ATMT）。 OSCAR非常快速，并且与其他策略（例如在酵母2中使用吉布森装配）相比较。 OSCAR方法成功应用于几种真菌菌根霉菌。这些物种包括: 镰孢镰孢 （未发表）， 大丽花 ， e 3 ，，ica ica ica ica ii ii ii ii ii ii um um um um um。。。。。 SP。 Vasinfectum 6 ，Pestalotiopsis microspora 7 ，Colletotrichum higginsianum 8 和Dothistroma septosporum 9和Sarocladium zeae （未发表） 。 该方案提供了包括引物设计，侧翼PCR扩增，OSCAR BP反应，缺失构建结构确认，转化的方法的逐步说明土壤杆菌的离子与构建物接着基于ATMT将缺失构建体转移到真菌细胞中，最后将真菌缺失突变体与具有异位整合的缺失构建体的突变体区分开。

<table><tbody><tr><td>FungiDB</td><td></td><td></td><td>Database/ http://fungidb.org/fungidb/</td></tr><tr><td>IDT PrimerQuest</td><td>IDT</td><td></td><td>Primer design online software/ http://www.idtdna.com/Primerquest/Home/Index</td></tr><tr><td>Microsoft Word</td><td></td><td></td><td>Sequence file manipulation</td></tr><tr><td>Low Na LB Spec 100 medium</td><td></td><td></td><td>&lt;em&gt;E. coli&lt;/em&gt; transformant selection, composition: 1% tryptone, 0.05% NaCl, 0.5% yeast extract, 1.5% agar if for solid medium</td></tr><tr><td>Co-cultivation medium</td><td></td><td></td><td>ATMT transformation induction (Reference 12)</td></tr><tr><td>Aspergillus minimal medium with Hygromycin</td><td></td><td></td><td>Fungal transformant selection</td></tr><tr><td>PDA medium</td><td>Acumedia</td><td>7149A</td><td>Single spore slant tubes</td></tr><tr><td>PDA-Hyg-Kan medium</td><td></td><td></td><td>Fungal ransformant isolation, PDA containing 150 &amp;mu;g/mL hygromycin B and 100 &amp;mu;g/mL Kanamycin</td></tr><tr><td>Glass beads</td><td>Genlantis</td><td>C400100</td><td>Plate spreading</td></tr><tr><td>Nitrocellulose filters (47 mm)</td><td>Fisher</td><td>09-719-555</td><td>Co-culturing for ATMT</td></tr><tr><td>Various centifuge tubes</td><td></td><td></td><td>multiple preps</td></tr><tr><td>Petri plates (various)</td><td></td><td></td><td>Culturing of bacteria and Fungi</td></tr><tr><td>pA-Hyg OSCAR</td><td>Addgene</td><td>29640</td><td>Selectable marker vector</td></tr><tr><td>pOSCAR</td><td>Addgene</td><td>29639</td><td>Assembly vector</td></tr><tr><td>DH5&amp;#945; One Shot Competent &lt;em&gt;E. coli &lt;/em&gt;cells</td><td>Life Technologies&amp;nbsp;</td><td>12297-016</td><td>BP reaction transformation</td></tr><tr><td>ccdB survival E. coli cells</td><td>Life Technologies&amp;nbsp;</td><td>A10460</td><td>Maintenance of pOSCAR</td></tr><tr><td>Wooden transfer sticks</td><td></td><td></td><td>Colony streaking</td></tr><tr><td>Toothpicks</td><td></td><td></td><td>Colony picking</td></tr><tr><td>Microcentrifuge</td><td></td><td></td><td>Pelleting Bacteria, &lt;em&gt;etc.&lt;/em&gt;</td></tr><tr><td>Preparative centrifuge</td><td></td><td></td><td>Fungal spore collection</td></tr><tr><td>Dissecting microscope</td><td></td><td></td><td>Single spore isolation</td></tr><tr><td>Automated Cell Counter</td><td></td><td></td><td>Spore suspension calculation</td></tr><tr><td>Compound microscope</td><td></td><td></td><td>Hemocytometer cell counting</td></tr><tr><td>QIAquick PCR Purification Kit&amp;nbsp;</td><td>Qiagen</td><td>28104</td><td>PCR gene flank produict purification</td></tr><tr><td>TaKaRa LA &lt;em&gt;Taq&lt;/em&gt;&amp;nbsp;</td><td>Takara Bio USA</td><td>RR002A</td><td>Hi Fidelity &lt;em&gt;taq &lt;/em&gt;polymerase for OSCAR flank generation</td></tr><tr><td>Hygromycin B</td><td>InvivoGen</td><td>ant-hg-5</td><td></td></tr><tr><td>Spectinomycin</td><td>Sigma</td><td>22189-32-8</td><td></td></tr><tr><td>Cefotaxime</td><td>TCI America</td><td>C2224</td><td></td></tr><tr><td>Kanamycin</td><td></td><td>11815032</td><td></td></tr><tr><td>Moxalactam</td><td>Sigma-Aldrich</td><td>43963</td><td></td></tr><tr><td>GelRed</td><td>&amp;nbsp;Phenix Research Products</td><td>RGB-4103</td><td>Post staining agarose gels</td></tr><tr><td>Qiagen QIAquick PCR Purification Kit (Cat. No. 28104)&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>(OneShot_ Mach1TM T1R or One Shot_ OmniMAX&amp;trade; 2 T1R from Invitrogen)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>C862003</td><td></td></tr><tr><td>Gateway BP Clonase II Enzyme mix</td><td>Thermo Fisher Scientific</td><td>11789020</td><td>Used to assemble deletion construct in pOSAR</td></tr></tbody></table>

rapid deletion production in fungi via agrobacterium mediated transformation of oscar deletion constructs

精确删除感兴趣的基因，同时使基因组的其余部分保持不变，为确定特定基因在活体中的功能提供了理想的产物。在本协议中，描述了精确快速删除质粒构建的OSCAR方法。 OSCAR依赖于进行单次重组酶反应的克隆系统，其中包含纯化的PCR扩增的目的基因的5&#39;和3&#39;侧翼和两个质粒pA-Hyg OSCAR（标记载体）和pOSCAR（组装体向量）。正确组装的缺失载体的确认通过限制性消化作图进行测序。然后使用根癌土壤杆菌介导将缺失构建体引入真菌孢子（简称ATMT）。最后，描述PCR测定以确定是否通过同源或非同源重组整合的缺失构建体，表明基因缺失或异位整合。这种方法已被成功地用于删除大丽花轮枝 菌属和其他物种中的尖角 镰孢属（Fusarium v​​erticillioides）中的许多基因。

在单个反应中，OSCAR方法产生含有围绕选择性标记盒的待缺失的靶基因的侧翼的质粒。使用OSCAR生成删除结构非常有效。然而，该系统可以产生含有一些但不是全部三个片段（两个基因侧翼和可选择标记）的部分构建体。通常，大多数大肠杆菌转化体含有正确的OSCAR构建体。例如， 图2描述了大丽轮枝菌基因VDAG_06812的OSCAR缺失构建体。在这种情况下...

Watch this Scientific Journal Video about Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs at JoVE.com

Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs

通过同源重组产生的基因缺失突变体是基因功能研究的黄金标准。描述了用于快速产生缺失构建体的OSCAR（农杆菌 - 重组 - 质粒的一步构建）方法。 土壤杆菌介导的真菌转化如下。最后，提出了基于真菌转化体基因缺失的基于PCR的确认方法。

真菌快速缺失生产<em&gt;农杆菌</em&gt;介入OSCAR删除构造的转换

Precise deletion of gene(s) of interest, while leaving the rest of the genome unchanged, provides the ideal product to determine that particular ...

rapid-deletion-production-fungi-via-agrobacterium-mediated

Research

JoVE Journal

Genetics

Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs

9.9K 次观看.  NPRC, USDA-ARS. 通过同源重组产生的基因缺失突变体是基因功能研究的黄金标准。描述了用于快速产生缺失构建体的OSCAR（农杆菌 - 重组 - 质粒的一步构建）方法。 土壤杆菌介导的真菌转化如下。最后，提出了基于真菌转化体基因缺失的基于PCR的确认方法。 

视频: Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic characteristics, this unconventional yeast is not only a good model for the study of the fundamental nature of fungal differentiation but is also a promising microbial platform for biochemical production and various biotechnological applications, which require extensive genetic manipulations. However, genetic manipulations of Y. lipolytica have been limited due to the lack of an efficient and stable genetic transformation system as well as very high rates of non-homologous recombination that can be mainly attributed to the KU70 gene. Here, we report an easy and rapid protocol for the efficient genetic transformation and for gene deletion in Y. lipolytica Po1g. First, a protocol for the efficient transformation of exogenous DNA into Y. lipolytica Po1g was established. Second, to achieve the enhanced double-crossover homologous recombination rate for further deletion of target genes, the KU70 gene was deleted by transforming a disruption cassette carrying 1 kb homology arms. Third, to demonstrate the enhanced gene deletion efficiency after deletion of the KU70 gene, we individually deleted 11 target genes encoding alcohol dehydrogenase and alcohol oxidase using the same procedures on the KU70 knockout platform strain. It was observed that the rate of precise homologous recombination increased substantially from less than 0.5% for deletion of the KU70 gene in Po1g to 33%-71% for the single gene deletion of the 11 target genes in Po1g KU70&#916;. A replicative plasmid carrying the hygromycin B resistance marker and the Cre/LoxP system was constructed, and the selection marker gene in the yeast knockout strains was eventually removed by expression of Cre recombinase to facilitate multiple rounds of targeted genetic manipulations. The resulting single-gene deletion mutants have potential applications in biofuel and biochemical production.

We herein report methods on the molecular genetic manipulation of the Yarrowia lipolytica Po1g strain for improved gene deletion efficiency. The resulting engineered Y. lipolytica strains have potential applications in biofuel and biochemical production.

可再生生物燃料及生化生产的非常规酵母基因工程

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic ...

科研

遗传学

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell&#39;s DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

We describe a robust gene replacement strategy to genetically manipulate the smut fungus Ustilago maydis. This protocol explains how to generate deletion mutants to investigate infection phenotypes. It can be extended to modify genes in any desired way, e.g., by adding a sequence encoding a fluorescent protein tag.

植物病原体的遗传操作<em&gt;玉米黑粉菌</em&gt;研究真菌生物学和植物微生物相互作用

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

This article describes a detailed methodology for random mutagenesis of a target gene in fission yeast. As an example, we target rpt4+, which encodes a subunit of the 19S proteasome, and screen for mutations that destabilize heterochromatin.

基因靶向随机诱变在裂变酵母中选择染色质稳定蛋白酶突变体

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol for transferring gene deletion mutations from one Escherichia coli strain to another by using generalized transduction with the bacteriophage P1. This method requires that the mutation be selectable (e.g., based on gene disruptions using antibiotic cassette insertions). Such antibiotic cassettes can be mobilized from a donor strain and introduced into a recipient strain of interest to quickly and easily generate a gene deletion mutant. The antibiotic cassette can be designed to include flippase recognition sites that allow the excision of the cassette by a site-specific recombinase to produce a clean knock-out with only a ~100-base-pair-long scar sequence in the genome. We demonstrate the protocol by knocking out the tamA gene encoding an assembly factor involved in autotransporter biogenesis and test the effect of this knock-out on the biogenesis and function of two trimeric autotransporter adhesins. Though gene deletion by P1 transduction has its limitations, the ease and speed of its implementation make it an attractive alternative to other methods of gene deletion.

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

Here we present a protocol for the use of pre-existing antibiotic resistance-cassette deletion constructs as a basis for making deletion mutants in other E. coli strains. Such deletion mutations can be mobilized and inserted into the corresponding locus of a recipient strain using P1 bacteriophage transduction.

可抽税抗生素耐药盒 P1 转导制备大肠杆菌中的基因缺失

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol ...

Direct Liquid-Culture Screening for Evaluating the Production of Heterologous Proteins Using an Auxotrophic Mutant of Aspergillus Oryzae

Aspergillus oryzae, a filamentous fungus, is one of the most widely used hosts for industrial applications including large-scale production of proteins. A polyethylene glycol (PEG)-mediated protoplast transformation method is generally used for the introduction of heterologous genes into A. oryzae. The conventional method typically requires three weeks for the screening of favorable transformants. Here, a new technique, the direct liquid-culture (DLC) screening method, is introduced which reduces the screening time to six days in a 200 mL flask format or to 10 days in a 24 well microplate format. The DLC screening method ensures the acquisition of positive transformants and evaluation of the secretory production of heterologous proteins in a single step, unlike the conventional screening method where two separate steps are required for the same. The protocol for PEG-mediated protoplast transformation of A. oryzae is described, which consists of five steps: preparation of fresh spore suspension, preculture, preparation of protoplasts, introduction of DNA, and DLC screening. For successful results in DLC screening, it is critical to use a nutrient-rich medium with optimized osmotic pressure. The protocol should further popularize the use of A. oryzae as a host of choice in the industrial production of proteins.

<meta charset="utf8"></head><body>直接液体培养筛选，用于评估使用米曲霉营养缺陷型突变体生产异源蛋白

A direct liquid-culture (DLC) screening method has been developed which significantly reduces the time required for polyethylene glycol (PEG)-mediated protoplast transformation of the filamentous fungus, Aspergillus oryzae, when employed for evaluation of the secretory production of heterologous proteins. This method dramatically increases the throughput of the evaluation protocol.

直接液体培养筛选，用于评估使用米曲霉营养缺陷型突变体生产异源蛋白

Aspergillus oryzae, a filamentous fungus, is one of the most widely used hosts for industrial applications including large-scale production of proteins. A ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

绿色怪物承载多基因缺失的酵母菌株的产生过程

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

生物学

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

全基因组基因缺失<em&gt;链球菌血统</em通过高通量PCR

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

基因缺失在通过CRISPR / Cas9哺乳动物细胞系生成

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.

定向进化法<em&gt;酿酒酵母</em&gt;:突变体库的创建和筛选

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that ...

真菌快速缺失生产

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可再生生物燃料及生化生产的非常规酵母基因工程

植物病原体的遗传操作

基因靶向随机诱变在裂变酵母中选择染色质稳定蛋白酶突变体

可抽税抗生素耐药盒 P1 转导制备大肠杆菌中的基因缺失

直接液体培养筛选，用于评估使用米曲霉营养缺陷型突变体生产异源蛋白

直接液体培养筛选，用于评估使用米曲霉营养缺陷型突变体生产异源蛋白

绿色怪物承载多基因缺失的酵母菌株的产生过程

全基因组基因缺失

基因缺失在通过CRISPR / Cas9哺乳动物细胞系生成

定向进化法