Promoter Orientation of Prokaryotic Phase-variable Genes by PCR
采用PCR进行原核细胞可变基因启动子定位
发布: 2012年10月20日第2卷第20期 DOI: 10.21769/BioProtoc.274 浏览次数: 10174
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参见作者原研究论文
The authors used this protocol in:
Mar 2012
Abstract
One major mechanism of phase variable gene expression in prokaryotes is through inversion of the promoter element for a gene or operon. This protocol describes how to detect the promoter orientation of a phase-variable gene by PCR. This protocol, including primer design, is specific to detection of the promoter orientations of hyxR, a LuxR-like response regulator in Extraintestinal Pathogenic Escherichia coli (ExPEC) isolates (Bateman and Seed, 2012); however, this protocol can be generalized to other organisms and genes to discriminate prokaryotic promoter inversions by PCR through size discrimination of the amplification products. Expression of hyxR is regulated through bidirectional phase inversion of the upstream promoter region mediated by a member of the family of site-specific tyrosine recombinases called Fim-like recombinases. The recombinases recognize inverted DNA repeat sequences flanking the promoter and produce a genomic rearrangement, orientating the promoter in favor or disfavor of gene expression.
Materials and Reagents
- Escherichia coli (E. coli) isolate UTI893
- Sterile distilled, deionized water (diH2O)
- Agarose, molecular biology grade, standard sieve
- Ethidium Bromide, 1 mg/ml in distilled, deionized water (diH2O) (or other agent to visualize DNA)
- Taq polymerase (1 U/μl) with 10x NH4 buffer (APEX Bioresearch Products, catalog number: 42-409 )
- 10 mM dNTP mix, PCR grade (Life Technologies, catalog number: 18427-013 )
- Tryptone
- Yeast extract
- NaCl
- Tris base
- Boric acid
- EDTA (pH 8.0)
- SDS
- Glycerol
- Xylene cyanol
- Bromophenol blue
- hyxR phase-specific primers, 100 μM stock solution [Integrated DNA Technologies (IDT)]
5’ – ACTGATAATAACCAGAGGCTTCTT – 3’
5’ – CAGTGATTAACTTTCGAACATATTG – 3’
5’ – GCGAAAGTTAATCACTGGTATGACC – 3’ - Tris-Borate-EDTA (TBE) (10x stock) (see Recipes)
- 10x DNA Loading Dye (see Recipes)
- Luria-Bertani broth (LB) culture medium (Sigma-Aldrich, catalog number: L3022-250G (see Recipes)
- 2% TBE agarose gel with EtBr (see Recipes)
- 20 ml 10x DNA Loading dye6 (xylene cyanol/bromophenol blue) (see Recipes)
Equipment
- 37 °C Incubator for bacteria with aeration
- Thermal cycler (Bio-Rad Laboratories)
- Gel electrophoresis system (Owl Separation Systems)
- UV Transilluminator with photo documentation (Bio-Rad Laboratories)
Procedure
文章信息
版权信息
© 2012 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Bateman, S. L. and Seed, P. (2012). Promoter Orientation of Prokaryotic Phase-variable Genes by PCR. Bio-protocol 2(20): e274. DOI: 10.21769/BioProtoc.274.
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分类
微生物学 > 微生物遗传学 > DNA
分子生物学 > DNA > PCR
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