Precisely determine the regulator-protected DNA sequences by DNase I Footprinting assay
A DNase foot printing assay is often used to detect DNA-protein interaction employing the fact that a protein bound to DNA will often protect the region of DNA from enzymatic cleavage, e.g. DNase I digestion. This makes it possible to locate the precise protein binding site on a particular DNA molecule. The method uses DNase I to cut the one end-labeled DNA, followed by gel electrophoresis to detect the resulting cleavage pattern.
Previously, the probe is usually labeled with radioactive isotopes (e.g. 32P), which is dangerous and environmentally hostile. In addition, the following sequencing PAGE electrophoresis is difficult to manage and time-consuming. Now, Shanghai Tolo Biotech has developed a new platform, employing the Fluorescent labelling method and ABI sequencer, for the DNase I Footprinting assay.
1. The Advantage of Fluorescent Labelling Method
1) Relative to the radioactive method, fluorescent probes are much after and environmentally friendly.
2) Fluorescent labelling is time-saving, highly repeatable and has much higher sensitivity.
2. Experimental Procedures
(a) Sample quality control;
(b) EMSA verification;
(c) DNase I digestion;
(d) ABI sequencer analysis;
(e) Data analysis and figure preparation;
(f) Experiment Reports.
(1) If the samples failed in the EMSA verification stage, ToloBio only charges the QC fee.
(2) We promise that all experimental data provided by ToloBio are real and repeatable.
(3) ToloBio will work together with you to solve any problem during the data publication, e.g. the referees may require more experiments or merely a detailed explanation of the experiments.
3. More Info
● This service is more suitable for reseachers who have employed the EMSA to prove that the protein can bind the target DNA. Then, DNase I Footprinting assay can be employed to precisely determine the protein binding DNA sequences.
● Customers may need to provide purified proteins, cloned DNA samples (target DNA should be between 100-400 bps), the target DNA sequences, EMSA figures and experimental conditions to favour the following DNase I Footprinting assay.
● ToloBio provides other services, including plasmid construction, protein expression and purification, EMSA. On the basis of DNase I Footprinting assay, ToloBio provides further services including both in vitro (e.g. site-directed mutagenesis combining EMSA/DNase I Footprinting assay) and in vivo (e.g. site-directed mutagenesis combing a reporter assay) experiments for a better understanding of the binding and regulation.
4. Recently published papers using ToloBio DNase I Footprinting assay techniques (Partial)
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 mediterranei. Microbial Cell Factories17:14; DOI: 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 coelicolor. Frontiers 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 mediterranei. Applied 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.