Name: essential components of borreliella (borrelia) burgdorferi in vitro transcription assays
Uploaded: 2022-07-22T14:40:00
Description: Watch this Scientific Journal Video about Essential Components of Borreliella Borrelia burgdorferi In Vitro Transcription Assays at JoVE.com

这项工作得到了克赖顿大学健康科学战略投资基金教师发展补助金的支持。B. burgdorferi RpoC-His10X菌株由蒙大拿大学的D. Scott Samuels博士慷慨提供。携带编码麦芽糖结合蛋白标记rpoD等位基因的pMAL-C5X质粒的大肠杆菌菌株由NIH落基山实验室的Frank Gherardini博士友好提供。

1. RNA聚合酶的纯化和RNA聚合酶原液的制备
<ol><li>使用先前描述的细胞收集方案 28，29 从 2-4 L 伯氏疏螺旋体 RpoC-His10X 中培养的 BSKII 培养基中培养的细胞沉淀 28，29。在500mL离心瓶中以10，000×g在4°C下离心30分钟，倒出上清液。</li>
	<li>将细胞重悬于30 mL冰冷的HN缓冲液（10 mM HEPES，10 mM NaCl，pH 8.0）中。使?...

<ol>
	<li>Brisson, D., Drecktrah, D., Eggers, C. H., Samuels, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+Borrelia+burgdorferi.">Genetics of Borrelia burgdorferi.</a> Annual Review of Genetics. 46, 515-536 (2012).</li><li>Kingry, L. 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=Surveillance+for+and+discovery+of+Borrelia+Species+in+US+patients+suspected+of+tickborne+illness.">Surveillance for and discovery of Borrelia Species in US patients suspected of tickborne illness.</a> Clinical Infectious Diseases. 66 (12), 1864-1871 (2018).</li><li>Cutler, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relapsing+fever+Borreliae:+A+global+review.">Relapsing fever Borreliae: A global review.</a> Clinics in Laboratory Medicine. 35 (4), 847-865 (2015).</li><li>Barbour, A. G., Hayes, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+Borrelia+species.">Biology of Borrelia species.</a> Microbiological Reviews. 50 (4), 381-400 (1986).</li><li>Tilly, K., Rosa, P. A., Stewart, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+infection+with+Borrelia+burgdorferi.">Biology of infection with Borrelia burgdorferi.</a> Infectious Disease Clinics of North America. 22 (2), 217-234 (2008).</li><li>Radolf, J. D., Caimano, M. J., Stevenson, B., Hu, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+ticks,+mice+and+men:+understanding+the+dual-host+lifestyle+of+Lyme+disease+spirochaetes.">Of ticks, mice and men: understanding the dual-host lifestyle of Lyme disease spirochaetes.</a> Nature Reviews Microbiology. 10 (2), 87-99 (2012).</li><li>Caimano, M. J., Drecktrah, D., Kung, F., Samuels, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+the+Lyme+disease+spirochete+with+its+tick+vector.">Interaction of the Lyme disease spirochete with its tick vector.</a> Cellular Microbiology. 18 (7), 919-927 (2016).</li><li>Dulebohn, D. P., Richards, C. L., Su, H., Lawrence, K. A., Gherardini, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Weak+organic+acids+decrease+Borrelia+burgdorferi+cytoplasmic+pH,+eliciting+an+acid+stress+response+and+impacting+RpoN-+and+RpoS-dependent+gene+expression.">Weak organic acids decrease Borrelia burgdorferi cytoplasmic pH, eliciting an acid stress response and impacting RpoN- and RpoS-dependent gene expression.</a> Frontiers in Microbiology. 8, 1734 (2017).</li><li>Bontemps-Gallo, S., Lawrence, K., Gherardini, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+different+virulence-related+regulatory+pathways+in+Borrelia+burgdorferi+are+directly+affected+by+osmotic+fluxes+in+the+blood+meal+of+feeding+ixodes+ticks.">Two different virulence-related regulatory pathways in Borrelia burgdorferi are directly affected by osmotic fluxes in the blood meal of feeding ixodes ticks.</a> PLoS Pathogens. 12 (8), 1005791 (2016).</li><li>Bugrysheva, J. V., 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=Characterization+of+the+RelBbu+Regulon+in+Borrelia+burgdorferi+reveals+modulation+of+glycerol+metabolism+by+(p)ppGpp.">Characterization of the RelBbu Regulon in Borrelia burgdorferi reveals modulation of glycerol metabolism by (p)ppGpp.</a> PLoS One. 10 (2), 0118063 (2015).</li><li>Carroll, J. A., Garon, C. F., Schwan, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+environmental+pH+on+membrane+proteins+in+Borrelia+burgdorferi.">Effects of environmental pH on membrane proteins in Borrelia burgdorferi.</a> Infection and Immunity. 67 (7), 3181-3187 (1999).</li><li>Drecktrah, D., 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=The+Borrelia+burgdorferi+RelA/SpoT+homolog+and+stringent+response+regulate+survival+in+the+tick+vector+and+global+gene+expression+during+starvation.">The Borrelia burgdorferi RelA/SpoT homolog and stringent response regulate survival in the tick vector and global gene expression during starvation.</a> PLoS Pathogens. 11 (9), 1005160 (2015).</li><li>Hyde, J. A., Trzeciakowski, J. P., Skare, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Borrelia+burgdorferi+alters+its+gene+expression+and+antigenic+profile+in+response+to+CO2+levels.">Borrelia burgdorferi alters its gene expression and antigenic profile in response to CO2 levels.</a> Journal of Bacteriology. 189 (2), 437-445 (2007).</li><li>Lin, Y. H., Chen, Y., Smith, T. C., Karna, S. L. R., Seshu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-chain+fatty+acids+alter+metabolic+and+virulence+attributes+of+Borrelia+burgdorferi.">Short-chain fatty acids alter metabolic and virulence attributes of Borrelia burgdorferi.</a> Infection and Immunity. 86 (9), 00217-00218 (2018).</li><li>Seshu, J., Boylan, J. A., Gherardini, F. C., Skare, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissolved+oxygen+levels+alter+gene+expression+and+antigen+profiles+in+Borrelia+burgdorferi.">Dissolved oxygen levels alter gene expression and antigen profiles in Borrelia burgdorferi.</a> Infection and Immunity. 72 (3), 1580-1586 (2004).</li><li>Stevenson, B., Schwan, T. G., Rosa, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-related+differential+expression+of+antigens+in+the+Lyme+disease+spirochete,+Borrelia+burgdorferi.">Temperature-related differential expression of antigens in the Lyme disease spirochete, Borrelia burgdorferi.</a> Infection and Immunity. 63 (11), 4535-4539 (1995).</li><li>Troxell, B., 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=Manganese+and+zinc+regulate+virulence+determinants+in+Borrelia+burgdorferi.">Manganese and zinc regulate virulence determinants in Borrelia burgdorferi.</a> Infection and Immunity. 81 (8), 2743-2752 (2013).</li><li>Yang, X., 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=Interdependence+of+environmental+factors+influencing+reciprocal+patterns+of+gene+expression+in+virulent+Borrelia+burgdorferi.">Interdependence of environmental factors influencing reciprocal patterns of gene expression in virulent Borrelia burgdorferi.</a> Molecular Microbiology. 37 (6), 1470-1479 (2000).</li><li>Samuels, D. S., 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=Gene+regulation+and+transcriptomics.">Gene regulation and transcriptomics.</a> Current Issues in Molecular Biology. 42, 223-266 (2021).</li><li>Fraser, C. M., 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=Genomic+sequence+of+a+Lyme+disease+spirochaete,+Borrelia+burgdorferi.">Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi.</a> Nature. 390 (6660), 580-586 (1997).</li><li>Iyer, R., Schwartz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray-based+comparative+genomic+and+transcriptome+analysis+of+Borrelia+burgdorferi.">Microarray-based comparative genomic and transcriptome analysis of Borrelia burgdorferi.</a> Microarrays. 5 (2), 9 (2016).</li><li>Iyer, R., 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=Stage-specific+global+alterations+in+the+transcriptomes+of+Lyme+disease+spirochetes+during+tick+feeding+and+following+mammalian+host+adaptation.">Stage-specific global alterations in the transcriptomes of Lyme disease spirochetes during tick feeding and following mammalian host adaptation.</a> Molecular Microbiology. 95 (3), 509-538 (2015).</li><li>Boyle, W. K., 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=DksA-dependent+regulation+of+RpoS+contributes+to+Borrelia+burgdorferi+tick-borne+transmission+and+mammalian+infectivity.">DksA-dependent regulation of RpoS contributes to Borrelia burgdorferi tick-borne transmission and mammalian infectivity.</a> PLoS Pathogen. 17 (2), 1009072 (2021).</li><li>Boyle, W. K., 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=Establishment+of+an+in+vitro+RNA+polymerase+transcription+system:+a+new+tool+to+study+transcriptional+activation+in+Borrelia+burgdorferi.">Establishment of an in vitro RNA polymerase transcription system: a new tool to study transcriptional activation in Borrelia burgdorferi.</a> Scientific Reports. 10 (1), 8246 (2020).</li><li>Silar, R., 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=Use+of+in+vitro+transcription+system+for+analysis+of+Corynebacterium+glutamicum+promoters+recognized+by+two+sigma+factors.">Use of in vitro transcription system for analysis of Corynebacterium glutamicum promoters recognized by two sigma factors.</a> Current Microbiology. 73 (3), 401-408 (2016).</li><li>Maciag, A., 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=In+vitro+transcription+profiling+of+the+sigmaS+subunit+of+bacterial+RNA+polymerase:+re-definition+of+the+sigmaS+regulon+and+identification+of+sigmaS-specific+promoter+sequence+elements.">In vitro transcription profiling of the sigmaS subunit of bacterial RNA polymerase: re-definition of the sigmaS regulon and identification of sigmaS-specific promoter sequence elements.</a> Nucleic Acids Research. 39 (13), 5338-5355 (2011).</li><li>Chang, A., 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=BRENDA,+the+ELIXIR+core+data+resource+in+2021:+New+developments+and+updates.">BRENDA, the ELIXIR core data resource in 2021: New developments and updates.</a> Nucleic Acids Research. 49, 498-508 (2021).</li><li>Samuels, D. S., Drecktrah, D., Hall, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+transformation+and+complementation.">Genetic transformation and complementation.</a> Methods in Molecular Biology. 1690, 183-200 (2018).</li><li>Hyde, J. A., Weening, E. H., Skare, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+transformation+of+Borrelia+burgdorferi.">Genetic transformation of Borrelia burgdorferi.</a> Current Protocols in Microbiology. 20, 1-17 (2011).</li><li>Ge, Y., Old, I. G., Saint Girons, I., Charon, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+a+large+Borrelia+burgdorferi+motility+operon+which+is+initiated+by+a+consensus+sigma70+promoter.">Molecular characterization of a large Borrelia burgdorferi motility operon which is initiated by a consensus sigma70 promoter.</a> Journal of Bacteriology. 179 (7), 2289-2299 (1997).</li><li>Ozaki, M., Wada, A., Fujita, N., Ishihama, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+phase-dependent+modification+of+RNA+polymerase+in+Escherichia+coli.">Growth phase-dependent modification of RNA polymerase in Escherichia coli.</a> Molecular Genetics and Genomics. 230 (1-2), 17-23 (1991).</li><li>Jasinski, D. L., Schwartz, C. T., Haque, F., Guo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+scale+purification+of+RNA+nanoparticles+by+preparative+ultracentrifugation.">Large scale purification of RNA nanoparticles by preparative ultracentrifugation.</a> Methods in Molecular Biology. 1297, 67-82 (2015).</li><li>Gierl, A., Zillig, W., Stetter, K. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+components+sigma+and+y+of+the+DNA-dependent+RNA+polymerase+of+Lactobacillus+curvatus+in+promotor+selection.">The role of the components sigma and y of the DNA-dependent RNA polymerase of Lactobacillus curvatus in promotor selection.</a> European Journal of Biochemistry. 125 (1), 41-47 (1982).</li><li>Stetter, K. O., Zillig, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+in+lactobacillaceae.+DNA-dependent+RNA+polymerase+from+Lactobacillus+curvatus.">Transcription in lactobacillaceae. DNA-dependent RNA polymerase from Lactobacillus curvatus.</a> European Journal of Biochemistry. 48 (2), 527-540 (1974).</li><li>Borbley, G., Schneider, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyanobacteria.">Cyanobacteria.</a> Methods in Enzymology. 167, 3 (1988).</li><li>Svetlov, V., Artsimovitch, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+bacterial+RNA+polymerase:+Tools+and+protocols.">Purification of bacterial RNA polymerase: Tools and protocols.</a> Methods in Molecular Biology. 1276, 13-29 (2015).</li><li>Shimada, T., Yamazaki, Y., Tanaka, K., Ishihama, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+whole+set+of+constitutive+promoters+recognized+by+RNA+polymerase+RpoD+holoenzyme+of+Escherichia+coli.">The whole set of constitutive promoters recognized by RNA polymerase RpoD holoenzyme of Escherichia coli.</a> PLoS One. 9 (3), 90447 (2014).</li><li>Daubendiek, S. L., Ryan, K., Kool, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rolling-circle+RNA+synthesis:+Circular+oligonucleotides+as+efficient+substrates+for+T7+RNA+polymerase.">Rolling-circle RNA synthesis: Circular oligonucleotides as efficient substrates for T7 RNA polymerase.</a> Journal of the American Chemical Society. 117 (29), 7818-7819 (1995).</li><li>Marras, S. A., Gold, B., Kramer, F. R., Smith, I., Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+measurement+of+in+vitro+transcription.">Real-time measurement of in vitro transcription.</a> Nucleic Acids Research. 32 (9), 72 (2004).</li><li>Chamberlin, M. J., Nierman, W. C., Wiggs, J., Neff, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+assay+for+bacterial+RNA+polymerases.">A quantitative assay for bacterial RNA polymerases.</a> Journal of Biological Chemistry. 254 (20), 10061-10069 (1979).</li></ol>

使用所提出的方案构建的体外转录测定最近用于研究转录因子在伯氏疏螺旋体中的作用，并可用于使用其他转录因子，sigma因子和分子23构建类似的实验。一旦获得来自伯氏疏螺旋体的活性RNA聚合酶并检测到其活性，就可以修改体外转录测定中的组分和条件。该测定具有高度的灵活性和可修改性。反应是模块化的，由冷冻原料制成，一旦选择了实验参数...

莱姆病和回归热是由疏螺旋体和疏螺旋体1，2，3 属的螺旋体病原体引起的。莱姆病是北美一种突出的媒介传播疾病，因此，伯氏疏螺旋体是研究螺旋体生物学的重要模式生物4，5。对B. burgdorferi转录调控机制的研究旨在更好地了解它在蜱载体和哺乳动物宿主之间循环时对环境变化的适应6，7。pH、温度、渗透压、营养可用性、短链脂肪酸、有机酸以及溶解氧和二氧化碳水平的变化调节了伯氏疏螺旋体在其节肢动物载体中存活和感染动物很重要的基因的表达8，9，10，11，12，13，14，15，16，17，18.将这些对刺激的反应与调节机制联系起来一直是伯氏疏螺旋体研究的一个重要方面19。
转录因子和西格玛因子控制执行细胞过程的基因的转录。莱姆病和回归热螺旋体含有一组相对稀疏的转录因子和替代西格玛因子。尽管如此，仍有复杂的转录变化指导伯氏双歧杆菌对环境的反应20，21，22。驱动伯氏双歧杆菌响应环境变化的转录变化的具体机制尚不清楚。体外转录测定是采用生化方法测定转录因子和西格玛因子的功能和调节机制的有力工具23，24，25，26。
最近建立了使用伯氏疏螺旋体RNA聚合酶的体外转录测定系统24。由于细菌通常具有独特的细胞生理学，不同物种和属的RNA聚合酶对酶纯化，酶储存和反应缓冲条件的反应不同27。B. burgdorferi在遗传上也与许多细菌物种相距甚远，其中RNA聚合酶已被研究20。酶制备的各个方面，如裂解、洗涤和洗脱缓冲液条件、储存缓冲液、体外转录反应缓冲液以及测定构建方法都会改变RNA聚合酶活性。在此，我们提供了一种用于纯化RNA聚合酶和sigma因子RpoD，生产线性双链DNA模板以及构建体外转录测定的方案，以促进使用该系统的实验室之间的可重复性。我们详细介绍了一个示例反应，以证明RpoD依赖性转录的线性范围，并讨论了该方法的局限性和替代方案。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.45 micron syringe filter</td>
			<td>Thermo Scientific</td>
			<td>726-2545</td>
			<td>Step 1.7 and 2.3</td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>MidSci</td>
			<td>C50B</td>
			<td>Step 1.3, and subsequent steps</td>
		</tr>
		<tr>
			<td>50 mL high-speed centrifuge tubes</td>
			<td>Thermo Scientific</td>
			<td>3119-0050PK</td>
			<td>Step 1.2</td>
		</tr>
		<tr>
			<td>500 mL Centrifuge bottles</td>
			<td>Thermo Scientific</td>
			<td>3120-9500PK</td>
			<td>Step 1.1</td>
		</tr>
		<tr>
			<td>B-PER and instruction manual</td>
			<td>Thermo Scientific</td>
			<td>78248</td>
			<td>Step 1.4 and 2.2</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Fisher Scientific</td>
			<td>10035-04-8</td>
			<td>Step 2.6</td>
		</tr>
		<tr>
			<td>Centrifugal filters 10 Kd cutoff</td>
			<td>Millipore Sigma</td>
			<td>UFC8010</td>
			<td>Step 1.11 and 2.11</td>
		</tr>
		<tr>
			<td>Cobalt resin and instruction manual</td>
			<td>Thermo Scientific</td>
			<td>89969</td>
			<td>Step 1.9</td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Acros Organics</td>
			<td>426380500</td>
			<td>Step 1.4 and subsequent steps</td>
		</tr>
		<tr>
			<td>Dnase (Nuclease)</td>
			<td>Millipore Sigma</td>
			<td>70746</td>
			<td>Step 1.4 and 2.2</td>
		</tr>
		<tr>
			<td>Factor Xa Protease&#160;</td>
			<td>Haematologic Technologies</td>
			<td>HCXA-0060</td>
			<td>Step 2.6</td>
		</tr>
		<tr>
			<td>GE Typhoon 5 Phosphoimager</td>
			<td>GE lifesciences</td>
			<td>Multiple</td>
			<td>Step 4.15</td>
		</tr>
		<tr>
			<td>Gel Imager</td>
			<td>Bio-Rad</td>
			<td>Mutiple</td>
			<td>Step 1.13 and subsequent protien quality check steps</td>
		</tr>
		<tr>
			<td>H2O for in vitro transcription</td>
			<td>Fisher Scientific</td>
			<td>7732-18-5</td>
			<td>Step 3.2 and 3.3</td>
		</tr>
		<tr>
			<td>high fidelity PCR kit</td>
			<td>New England Biolabs</td>
			<td>M0530S</td>
			<td>Step 3.1</td>
		</tr>
		<tr>
			<td>High-speed centrifuge</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Step 1.1, and subsequent steps</td>
		</tr>
		<tr>
			<td>HiTrap Heparin HP 5 x 1 mL</td>
			<td>Cytiva Life Sciences</td>
			<td>17040601</td>
			<td>Step 2.8</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>56750-100G</td>
			<td>Step 1.9</td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Thermo Scientific</td>
			<td>90082</td>
			<td>Step 1.4 and 2.2</td>
		</tr>
		<tr>
			<td>Magnesium chloride&#160;</td>
			<td>Fisher Scientific</td>
			<td>S25401</td>
			<td>Step 4.1</td>
		</tr>
		<tr>
			<td>Manganese chloride</td>
			<td>Fisher Scientific</td>
			<td>S25418</td>
			<td>Step 4.1</td>
		</tr>
		<tr>
			<td>Mini protean tetra cell</td>
			<td>Bio-Rad</td>
			<td>Mutiple</td>
			<td>Step 1.13 and subsequent protien quality check steps</td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Thermo Scientific</td>
			<td>85124</td>
			<td>Step 4.1</td>
		</tr>
		<tr>
			<td>NTP mixture</td>
			<td>Thermo Scientific</td>
			<td>R0481</td>
			<td>Step 4.1</td>
		</tr>
		<tr>
			<td>PCR purification kit</td>
			<td>Qiagen</td>
			<td>28506</td>
			<td>Step 3.2</td>
		</tr>
		<tr>
			<td>PCR tubes</td>
			<td>MidSci</td>
			<td>PR-PCR28ACF</td>
			<td>Step 1.12</td>
		</tr>
		<tr>
			<td>PD-10 Sephadex buffer exchange column and instruction manual</td>
			<td>Cytiva</td>
			<td>17085101</td>
			<td>Step 1.10 and 2.10 (gel filtration column)</td>
		</tr>
		<tr>
			<td>pMAL Protein Fusion 
			and Purification System Instruction manual</td>
			<td>New England Biolabs</td>
			<td>E8200S</td>
			<td>Step 2.1</td>
		</tr>
		<tr>
			<td>Polyacrylamide gels AnyKD</td>
			<td>Bio-Rad</td>
			<td>456-8125</td>
			<td>Step 1.13 and subsequent protien quality check steps</td>
		</tr>
		<tr>
			<td>Potassium glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>G1251</td>
			<td>Step 4.1</td>
		</tr>
		<tr>
			<td>Protease inhibitor</td>
			<td>Thermo Scientific</td>
			<td>78425</td>
			<td>Step 1.4 and 2.2</td>
		</tr>
		<tr>
			<td>Radiolabeled ATP</td>
			<td>Perkin Elmer</td>
			<td>BLU503H</td>
			<td>Step 4.2</td>
		</tr>
		<tr>
			<td>RNA Loading Dye, (2x)</td>
			<td>New England Biolabs</td>
			<td>B0363S</td>
			<td>Step 4.13</td>
		</tr>
		<tr>
			<td>Rnase inhibitor</td>
			<td>Thermo Scientific</td>
			<td>EO0381</td>
			<td>Step 4.1</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Biotek</td>
			<td>Mutiple</td>
			<td>Step 1.13 and subsequent protien quality check steps</td>
		</tr>
		<tr>
			<td>TBE-Urea gels 10 percent</td>
			<td>Bio-Rad</td>
			<td>4566033</td>
			<td>Step 4.14</td>
		</tr>
	</tbody>
</table>

essential components of borreliella (borrelia) burgdorferi in vitro transcription assays

伯氏疏螺旋体 是一种细菌病原体，代谢和基因组库有限。 伯氏双歧杆菌 在脊椎动物和蜱虫之间细胞外传播，并显着重塑其转录谱，以便在感染期间在不同的环境中存活。 伯氏疏螺旋体 研究的一个重点是清楚地了解细菌如何通过转录变化对其环境做出反应。 体外 转录测定允许对转录调节的基本机制进行生化解剖。在这里，我们提出了一个详细的方案，描述了 伯氏疏螺旋 体RNA聚合酶纯化和储存，西格玛因子纯化，DNA模板生成和 体外 转录测定。该协议描述了使用从 伯氏疏螺旋体 5A4 RpoC-HIS（5A4-RpoC）纯化的RNA聚合酶。5A4-RpoC是一种先前发表的菌株，在编码RNA聚合酶最大亚基的 rpoC 基因上携带10XHis标签。 体外 转录测定包括从菌株 5A4-RpoC 纯化的 RNA 聚合酶、管家西格玛因子 RpoD 的重组版本和 PCR 生成的双链 DNA 模板。虽然蛋白质纯化技术和组装体外转录测定的方法在概念上很好理解且相对常见，但RNA聚合酶的处理注意事项通常因生物 体 而异。此处介绍的方案设计用于伯 氏疏螺旋体 RNA聚合酶的酶学研究。该方法可用于测试转录因子、启动子和翻译后修饰对RNA聚合酶活性的作用。

在 体外 转录反应中，反应的限制步骤是西格玛因子介导的转录起始，转录活性应随西格玛因子的量线性增加。我们介绍了 体外 转录实验的制备，测试一系列RpoD浓度以及两种浓度的RNA聚合酶，以观察放射性标记的核苷酸掺入RNA产物所产生的变化信号。显示了制备RNA聚合酶、RpoD和双链DNA模板储备液的代表性结果。代表性的 体外 转录反应可作为可接受的RNA聚合酶活性水平的一?...

Watch this Scientific Journal Video about Essential Components of Borreliella Borrelia burgdorferi In Vitro Transcription Assays at JoVE.com

Essential Components of Borreliella Borrelia burgdorferi In Vitro Transcription Assays

体外转录测定可以破译伯氏疏螺旋体的转录调控机制。该协议描述了纯化伯氏疏螺旋体RNA聚合酶并进行体外转录反应的步骤。使用体外转录测定的实验方法需要对活性RNA聚合酶进行可靠的纯化和储存。

伯氏疏螺旋体（Borrelia）伯氏疏螺旋体外转录测定的基本成分

Borreliella burgdorferi is a bacterial pathogen with limited metabolic and genomic repertoires. B. burgdorferi transits extracellularly between ...

伯氏疏螺旋体的体外转录测定系统为研究人员研究控制RNA聚合酶酶活性的基因调控机制和因子提供了一种生化工具。该系统使我们能够测试转录因子、辅助因子、盐浓度和pH值如何影响伯氏疏螺旋体RNA聚合酶功能，这有助于我们全面了解基因调控机制。这种强大的技术还可以让我们筛选选择性抑制RNA聚合酶的药物，为开发治疗莱姆疏螺旋体病的新药打开大门。首先，从两到四升伯氏疏螺旋体RpoC-His10X中收集细胞沉淀，该细胞沉淀在微需氧环境中培养在BSKII培养基中，密度为每毫升2至4倍10常数。在500毫升离心瓶中以10，000倍G在4摄氏度下沉淀细胞30分钟，并弃去上清液。然后，将细胞重悬于30毫升冰冷的HN缓冲液中，重复离心步骤，并倒出上清液。工作时，将细胞、裂解物和纯化的蛋白质保持在 4 摄氏度或冰上，除非冷冻。通过向所使用的每个缓冲液中加入浓度为2毫摩尔的新鲜制备的二硫苏糖醇，将RNA聚合酶保持在还原环境中，并保持pH值8.0。使用商业细菌裂解试剂盒从伯氏疏螺旋体细胞沉淀中制备裂解物。将沉淀重悬于10至15毫升不含蛋白酶抑制剂的B-PER溶液中，并在冰上进行裂解五分钟。向裂解溶液中加入蛋白酶抑制剂混合物，并进行三轮超声处理。现在通过使用50毫升离心管离心和过滤来澄清细胞裂解物。向溶液中加入钴柱上样缓冲液，使总体积达到30毫升。通过以20， 000倍G离心30分钟来沉淀细胞碎片。之后，使用0.45微米注射器过滤器过滤上清液，并将裂解物和钴柱上样缓冲液稀释至最终稀释比例为1：10。按照制造商的说明，使用钴或镍树脂柱对澄清的细胞裂解物上清液进行亲和色谱。保存流通、洗涤和洗脱样品进行分析。按照制造商的说明，立即使用缓冲液交换柱将RNA聚合酶溶液缓冲溶液与储存缓冲溶液交换。然后，使用10千道尔顿截止离心过滤装置将RNA聚合酶浓缩至0.2至0.4毫克/毫升的浓度。通过分光光度计确定浓度并制备冷冻储备液。将 20 至 50 微升体积的 RNA 聚合酶等分装在 PCR 管中冷冻原液，并将 RNA 聚合酶储存在零下 80 摄氏度。准备工作表面，包括辐射工作台，以减少辐射污染。在冰上解冻新鲜的RNA聚合酶和RpoD储备液，并在移液前彻底混合所有解冻的冷冻储液根据实验要求在单独的NTP混合物中制备用于放射性标记核苷酸的主反应混合物。将实验组中的对照反应分配到PCR管中。将水、反应混合物、RNA 聚合酶和 RpoD 分配到指定的 PCR 管中后，通过移液混合试剂。将制备的材料转移到辐射工作台上，将α-32P标记的ATP加入NTP中，并通过轻轻移液混合。将NTP添加到含有体外转录反应混合物的试管中，并通过添加DNA模板开始体外转录反应。通过轻轻移液混合反应体积，并将试管在37摄氏度下在热循环仪或加热块中孵育五分钟。从热循环仪或加热块中取出反应，并通过向反应混合物中加入等体积含有50%甲酰胺的2X RNA上样染料来停止体外转录反应。通过在65摄氏度下在热循环仪或加热块中孵育反应五分钟来使酶变性。使用凝胶电泳，在180伏的10%至15%TBE尿素聚丙烯酰胺凝胶中分离体外转录的RNA，持续30至45分钟。去除含有未掺入放射性标记ATP的凝胶的任何部分后，将凝胶暴露于磷酸化筛选过夜，并使用磷酸化屏幕成像仪对放射性标记的RNA进行成像。SDS-PAGE显示对应于RNA聚合酶复合物的三种肽RpoC，RpoB和RpoA的三个条带。还观察到与MBP标记的重组伯氏疏螺旋体RpoD的大小相匹配的115千道尔顿肽。蛋白质混合物与因子XA蛋白酶的切割导致产生两种重要产物RpoD和MBP。伯氏疏螺旋体FLGB的启动子位点由PCR生成。RpoD浓度的两倍稀释导致累积的RNA产物水平降低。低RNA聚合酶浓度在RpoD浓度范围内产生的RNA产物较少。密度测量信号表明，在两个实验中，反应中存在的RpoD量之间存在线性关系。RNA聚合酶的酶活性在纯化、储存和实验过程中对多种因素都很敏感。新鲜的酶和试剂，以及良好的计划，为该方案提供了最大的成功机会。

Research

JoVE Journal

Immunology and Infection

Essential Components of Borreliella (Borrelia) burgdorferi In Vitro Transcription Assays

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

细胞和生物膜细胞外成分的定量并发

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified ...

科研

免疫与感染

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

量<em在体外</em&gt;激活的主人微血管内皮细胞在静态条件下测量的检测，以中性粒细胞粘附

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

高产净化<em&gt;恶性疟原虫</em&gt;裂殖子适用于Opsonizing抗体检测

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance

Vitronectin 在细菌黏附和血清耐药性中作用的实验研究

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

体外方法比较标的结合和CDC诱导治疗性抗体之间：应用在Biosimilarity分析

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

用于炎症反应的新内皮调节因子的筛选测定

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing

hiv 转录和拼接中延迟逆转剂的高通量体外评价

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune ...

Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana

Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana

模式触发的氧化爆裂和幼苗生长抑制测定在阿拉伯拉多普西斯塔利亚纳

Plants have evolved a robust immune system to perceive pathogens and protect against disease. This paper describes two assays that can be used to measure ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

活体细胞毒性试验研究肿瘤特异性 CD8+ t 细胞反应中的免疫调节

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems

内生菌与人类肠道微生物群使用体外浴发酵系统的相互作用分析

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, ...