Single cell-level detection and quantitation of leaky protein expression from any strongly regulated bacterial system

被引：6

作者：

Arora, Kanika ^{[1
]}

Mangale, Sachin S. ^{[2
]}

Guptasarma, Purnananda ^{[1
]}

机构：

[1] Indian Inst Sci Educ & Res IISER Mohali, Dept Biol Sci, CPSDE, Sas Nagar 140306, Punjab, India

[2] Wipro GE Healthcare Pvt Ltd, GE Healthcare Life Sci, Bangalore 560048, Karnataka, India

来源：

ANALYTICAL BIOCHEMISTRY | 2015年 / 484卷

关键词：

Negative regulation; Leaky repression; Gene expression; Fluorescence imaging; Bacterial nucleoid staining; Fluorescent HU; REPLICATION-INDUCED TRANSCRIPTION; ESCHERICHIA-COLI; STRATEGIES;

D O I：

10.1016/j.ab.2015.06.011

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Extremely low levels of "leaky" expression of genes in bacterial protein expression systems can severely curtail cell viability when expressed proteins are toxic. A general method for sensitive detection of such expression is lacking. Here, we present a method based on microscopic visualization of a fluorescent "reporter" protein (RFP-HU-A) constructed by fusing red fluorescent protein (RFP) to the N-terminus of a nucleoid-associated, histone-like DNA-binding protein, HU-A. Localization of RFP-HU-A within nucleoids facilitates detection, quantitation, and characterization of leaky expression at the single-cell level. (C) 2015 Elsevier Inc. All rights reserved.

引用

页码：180 / 182

页数：3

共 17 条

[1] Tightly regulated vectors for the cloning and expression of toxic genes [J].

Anthony, LC ;

Suzuki, H ;

Filutowicz, M .

JOURNAL OF MICROBIOLOGICAL METHODS, 2004, 58 (02) :243-250

[2]

Green MR, 2012, Cold spring harbor, Vfourth

[3] DOES REPLICATION-INDUCED TRANSCRIPTION REGULATE SYNTHESIS OF THE MYRIAD LOW COPY NUMBER PROTEINS OF ESCHERICHIA-COLI [J].

GUPTASARMA, P .

BIOESSAYS, 1995, 17 (11) :987-997

[4]

Khow Orawan, 2012, Asian Pacific Journal of Tropical Biomedicine, V2, P159, DOI 10.1016/S2221-1691(11)60213-X

[5] Protein blotting: a review [J].

Kurien, BT ;

Scofield, RH .

JOURNAL OF IMMUNOLOGICAL METHODS, 2003, 274 (1-2) :1-15

[6] Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I-1-I-2 regulatory elements [J].

Lutz, R ;

Bujard, H .

NUCLEIC ACIDS RESEARCH, 1997, 25 (06) :1203-1210

[7] H-NS OVER-EXPRESSION INDUCES AN ARTIFICIAL STATIONARY-PHASE BY SILENCING GLOBAL TRANSCRIPTION [J].

MCGOVERN, V ;

HIGGINS, NP ;

CHIZ, RS ;

JAWORSKI, A .

BIOCHIMIE, 1994, 76 (10-11) :1019-1029

[8] Bright monomeric red fluorescent protein with an extended fluorescence lifetime [J].

Merzlyak, Ekaterina M. ;

Goedhart, Joachim ;

Shcherbo, Dmitry ;

Bulina, Mariya E. ;

Shcheglov, Aleksandr S. ;

Fradkov, Arkady F. ;

Gaintzeva, Anna ;

Lukyanov, Konstantin A. ;

Lukyanov, Sergey ;

Gadella, Theodorus W. J. ;

Chudakov, Dmitriy M. .

NATURE METHODS, 2007, 4 (07) :555-557

[9] Replication-induced transcription of an autorepressed gene: The replication initiator gene of plasmid P1 [J].

Mukhopadhyay, S ;

Chattoraj, DK .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2000, 97 (13) :7142-7147

[10]

Neil B., 2012, MAT METHODS, V2, DOI [10.13070/mm.en.2.123., DOI 10.13070/MM.EN.2.123]

← 1 2 →