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DNase I足迹分析实验服务

DNase I Footprinting精确鉴定调控蛋白在DNA上的结合序列

服务简介

 DNase I footprinting assay(DNase I足迹分析实验)可以精确鉴定DNA结合蛋白(如转录调控蛋白)在DNA分子上的结合位点。转录调控蛋白通过与启动子区域结合来调控靶标基因的转录。目前,凝胶阻滞实验(EMSA)和DNase I足迹分析实验是重要的一套体外研究工具:凝胶阻滞实验可以确定转录因子是否与DNA直接结合,而DNase I足迹分析实验则可以精确鉴定其结合的DNA序列,从而帮助我们研究基因转录调控的机制。经典的DNase I足迹分析实验需要使用同位素进行标记,并在反应完成后通过DNA测序胶电泳来进行检测;该方法不仅危险(同位素)、费时;而且对操作者的技术要求比较高、成功率低。针对这种情况,上海吐露港生物科技目前推出利用“荧光标记”结合ABI测序仪的方法来进行DNase I足迹分析实验。 


服务优势

1. “荧光标记”法安全、无污染; 

2. “荧光标记”实验的速度快、重复性好、灵敏度高。 


优势说明:

(1)荧光标记法,由于可以进行DNA的精确定量,并可以在常规的实验台上面操作;同位素标记法只能通过放射性强度来估测,且操作时需要特别的保护装置,操作不便。因此,荧光标记法的可重复性要远远好于同位素操作。 
(2)原始的PAGE测序胶对操作者的技术要求很高,且条带的清晰度受到多种实验因素的影响;ABI测序仪的操作体系非常成熟,灵敏度高,重复性好。因此,荧光标记法更灵敏度,获得的保护区域更清晰,图片的质量更好。 


服务流程


流程说明:

(1)若客户的实验样品在质检步骤没有通过,公司仅收取少许的质检费用;

(2)公司对提供的任何DNase I footprinting实验图片,均保证数据的真实性和实验的可重复性;

(3)客户在利用本公司提供的图片进行论文发表时,若论文审稿人提出任何关于图片/实验方面的要求,吐露港生物承诺将帮助客户来一同解决(若属于客 户原来的实验范围内,公司将免费帮助客户解决问题)。


服务说明 

(1)该服务适用于:已经通过EMSA等实验证明转录调控蛋白可以结合DNA,但是需要通过DNase I足迹分析实验来进一步精确鉴定蛋白结合的DNA序列,以便研究其转录调控的机制。

(2)客户需要提供纯化的蛋白,克隆的启动子及其序列(启动子长度应介于100-300bp,并需要克隆至通用的T载体上,经测序无误后可用于实验),同时需要提供EMSA结果图及实验参数以便于接下来的DNase I足迹分析实验。 
(3)公司同时提供蛋白表达载体构建、蛋白表达条件优化、蛋白纯化、EMSA摸索等实验,但需另外收费。 
(4)在获得调控蛋白的结合位点之后,吐露港生物也提供进行进一步的体外(DNA结合位点的突变后进行EMSA或DNase I Footprinting实验)、体内(DNA结合位点突变结合reporter实验)实验来精确证明,但需另外收费。 


近年来使用ToloBio技术服务发表的部分论文

27, Tao Xu, Yang Wu, Zhiwei Lin, Ralph Bertram, Friedrich G?tz, Ying Zhang* and Di Qu*. Identification of Genes Controlled by the Essential YycFG Two-Component System Reveals a Role for Biofilm Modulation in Staphylococcus epidermidis. Front Microbiol. 2017 Apr 26;8:724. doi: 10.3389/fmicb.2017.00724.

26, JinPei Wang, WenMao Zhang, HongJun Chao and NingYi Zhou*. PnpM, a LysR-Type Transcriptional Regulator Activates the Hydroquinone Pathway in para-Nitrophenol Degradation in Pseudomonas sp. Strain WBC-3. Front. Microbiol., 14 

25, Chao Lei#, Jingzhi Wang#, Yuanyuan Liu, Xinqiang Liu, Guoping Zhao, Jin Wang*. A feedback regulatory model for RifQ-mediated repression of rifamycin export in Amycolatopsis mediterraneiMicrobial Cell Factories17:14DOI: 10.1186/s12934-018-0863-5

24, Zhendong Li#, Xinqiang Liu#, Jingzhi Wang, Ying Wang, Guosong Zheng, Yinhua Lu, Guoping Zhao, Jin Wang*. Insight into the molecular mechanism of the transcriptional regulation of amtB operon in Streptomyces coelicolorFrontiers in Microbiology 9: 264; doi: 10.3389/fmicb.2018.00264

23, Yanli Cao, Fanglin Zheng, Lei Wang, Guolei Zhao,Guanjun Chen, Weixin Zhang* and Weifeng Liu*. Rce1, a novel transcriptional repressor, regulates cellulasegene expression by antagonizing the transactivator Xyr1 in Trichoderma reesei. Mol Microbiol. (2017) Apr 5. doi: 10.1111/mmi.13685.

22, Chen Li#, XinQiang Liu#, Chao Lei,Han Yan, ZhiHui Shao, Ying Wang, GuoPing Zhao, Jin Wang*, XiaoMing Ding*. RifZ (AMED_0655) is apathway-specific regulator for rifamycin biosynthesis in Amycolatopsis mediterraneiApplied Environmental Microbiology (2017) DOI: 10.1128/AEM.03201-16.

21, Juan-Mei He#, Hong Zhu#, Guo-Song Zheng, Pan-Pan Liu, Jin Wang, Guo-Ping Zhao, Guo-Qiang Zhu*, Wei-Hong Jiang* and Yin-Hua Lu*. Direct Involvement of the Master Nitrogen Metabolism Regulator GlnR in Antibiotic Biosynthesis in Streptomyces. Journal of Biological Chemistry (2016) 291(51): jbc.M116.762476/ doi: 10.1074/jbc.M116.762476.

20, Chen L, Zou G, Wang J, Wang J, Liu R, Jiang Y, Zhao G, Zhou Z. Characterization of the Ca(2+) -responsive signaling pathway in regulating the expression and secretion of cellulases in Trichoderma reesei Rut-C30. Mol Microbiol. (2016) 100(3):560-75. doi: 10.1111/mmi.13334.

19, Kaifeng Wu, Hongmei Xu, Yuqiang Zheng, Libin Wang, Xuemei Zhang, and Yibing Yin. CpsR, a GntR family regulator, transcriptionally regulates capsular polysaccharide biosynthesis and governs bacterial virulence in Streptococcus pneumoniae. Sci Rep. (2016) 6: 29255.

18, Cen XF, Wang JZ, Zhao GP, Wang Y, Wang J. Molecular evidence for the coordination of nitrogen and carbon metabolisms, revealed by a study on the transcriptional regulation of the agl3EFG operon that encodes a putative carbohydrate transporter in Streptomyces coelicolor. Biochem Biophys Res Commun. (2016) 471(4):510-4. doi: 10.1016/j.bbrc.2016.02.044.

17, ZhiHui Shao#,WanXin Deng#, ShiYuan Li, JuanMei He,ShuangXi Ren, WeiRen Huang, YinHua Lu, GuoPing Zhao, ZhiMing Cai*, Jin Wang* (2015) GlnR-MediatedRegulation of ectABCD TranscriptionExpands the Role of the GlnR Regulon to Osmotic Stress Management. Journal of Bacteriology 197(19): 3041–3047.

16, He Huang, Cheng-Cheng Song, Zhi-Liang Yang, Yan Dong, Yao-Zhong Hu, and Feng Gao. Identification of the Replication Origins from Cyanothece ATCC 51142 and Their Interactions with the DnaA Protein: From In Silico to In Vitro Studies. Front Microbiol. (2015) 6:1370. doi: 10.3389/fmicb.2015.01370. 

15, Qiuxiang Cheng, Bo Cao, Fen Yao, Jinli Li, Zixin Deng, Delin You. Regulation of DNA phosphorothioate modifications by the transcriptional regulator DptB in Salmonella. Molecular Microbiology (2015) 97(6), 1186–1194

14, Tietao Wang, Meiru Si, Yunhong Song, Wenhan Zhu, Fen Gao, Yao Wang, Lei Zhang, Weipeng Zhang, Gehong Wei, ZhaoQing Luo, Xihui Shen. Type VI Secretion System Transports Zn2+ to Combat Multiple Stresses and Host Immunity. PLOS Pathogens (2015) DOI:10.1371/journal.ppat.1005020

13, Cao G, Howard ST, Zhang P, Wang X, Chen XL, Samten B, Pang X. EspR, a regulator of the ESX-1 secretion system in Mycobacterium tuberculosis, is directly regulated by the two-component systems MprAB and PhoPR. Microbiology. 2015 161(3):477-89. doi: 10.1099/mic.0.000023.
12, Yunhong Song, Xiao Xiao, Changfu Li, Tietao Wang, Ruoxi Zhao, Weipeng Zhang, Lei Zhang, Yao Wang, Xihui Shen. The dual transcriptional regulator RovM regulates the expression of AR3- and T6SS4-dependent acid survival systems in response to nutritional status in Yersinia pseudotuberculosis. Environmental Microbiology (2015) 17(11), 4631–4645

11, Wen-Mao Zhang, Jun-Jie Zhang, Xuan Jiang, Hongjun Chao and Ning-Yi Zhou. Transcriptional Activation of Multiple Operons Involved in para-Nitrophenol Degradation by Pseudomonas sp. Strain WBC-3 Appl. Environ. Microbiol. January 2015 vol. 81 no. 1 220-230

10, Lian Zhou, Xing-Yu Wang, Shuang Sun, Li-Chao Yang, Bo-Le Jiang, Ya-Wen He. Identification and characterization of naturally occurring DSF-family quorum sensing signal turnover system in the phytopathogen Xanthomonas. Environmental Microbiology (2015) 17(11), 4646–4658

9, Junling, Dun;Yawei, Zhao;Guosong, Zheng;Hong, Zhu;Lijun, Ruan;Wenfang, Wang;Mei, Ge;Weihong, Jiang;Yinhua, Lu. PapR6, a putative atypical response regulator, functions as a pathway-specific activator of pristinamycin II biosynthesis in Streptomyces pristinaespiralis. J Bacteriol. 2015 Feb;197(3):441-50. doi: 10.1128/JB.02312-14.

8, Shuang Qu, Qianjin Kang, Hang Wu, Lei Wang, Linquan Bai. Positive and negative regulation of GlnR in validamycin A biosynthesis by binding to different loci in promoter region. Appl Microbiol Biotechnol (2015) 99:4771–4783 DOI 10.1007/s00253-015-6437-0  

7, Lijuan Wang, Hongzhi Tang, Hao Yu, Yuxiang Yao, Ping Xu. An unusual repressor controls the expression of a crucial nicotine-degrading gene cluster in Pseudomonas putida?S16. Molecular Microbiology (2014) 91(6), 1252–1269

6, Ying Wang, Chen Li, Na Duan, Bin Li, Xiao-Ming Ding, Yu-Feng Yao, Jun Hu , Guo-Ping Zhao, Jin Wang. GlnR Negatively Regulates the Transcription of the Alanine Dehydrogenase Encoding Gene ald in Amycolatopsis mediterranei U32 under Nitrogen Limited Conditions via Specific Binding to Its Major Transcription Initiation Site
PLoS ONE 2014; 9(8): e104811. doi:10. 1371/journal.pone.0104811

5, Zhongchao Zhao, Zhengfu Zhou, Liang Li, Xianyi Xian, Xiubin Ke, Ming Chen and    Yuxiu Zhang. A copper-responsive gene cluster is required for copper homeostasis and contributes to oxidative resistance in Deinococcus radiodurans R1. Mol. BioSyst., 2014,10, 2607-2616

4, J. Zhang, J. Xiao, Y*. Zhang, S. Cui, Q. Liu, Q. Wang, H. Wu, Y. Zhang. A new target for the old regulator: H-NS suppress T6SS secretory protein EvpP, the major virulence factor in the fish pathogen Edwardsiella tarda. Letters in Applied Microbiology (2014) 59, 557--564 

3, Z Yu,H Zhu,G Zheng,W Jiang,Y Lu. A genome-wide transcriptomic analysis reveals diverse roles of the two-component system DraR-K in the physiological and morphological differentiation of Streptomyces coelicolor. Appl Microbiol Biotechnol (2014) 98:9351–9363 DOI 10.1007/s00253-014-6102-z  

2, Ying Wang, Jing-Zhi Wang, Zhi-Hui Shao, Hua Yuan, Yin-Hua Lu, Wei-Hong Jiang, Guo-Ping Zhao, Jin Wang. Three of four GlnR binding sites are essential for GlnR-mediated activation of transcription of the Amycolatopsis mediterranei nas operon. J Bacteriol. 2013 Jun;195(11):2595-602. doi: 10.1128/JB.00182-13.

1, Ying Wang, Xu-Feng Cen, Guo-Ping Zhao, Jin Wang. Characterization of a new GlnR binding box in the promoter of amtB in Streptomyces coelicolor inferred a PhoP/GlnR competitive binding mechanism for transcriptional regulation of amtB. J Bacteriol. 2012 Oct;194(19):5237-44. doi: 10.1128/JB.00989-12.

                                                                                          

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