Silencing of Antibiotic Resistance in E. coli with Engineered Phage Bearing Small Regulatory RNAs

Libis, Vincent; Bernheim, Aude; Basier, Clovis; Jaramillo, Alfonso; Deyell, Matthew; Aghoghogbe, Idonnya; Atanaskovic, Iva; Bencherif, Amel Camélia; Benony, Marguerite; Koutsoubelis, Nicolas; Löchner, Anne; Marinkovic, Zoran S.; Zahra, Sarah; Zegman, Yonatan; Lindner, Ariel B.; Wintermute, Edwin H.

doi:10.1021/sb500033d

Cited by 35 publications

(22 citation statements)

References 9 publications

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“…The addition of ampicillin, to select for only bacteria carrying the phagemid, reduced chloramphenicol survival to undetectable levels. This is consistent with earlier work showing that escape from phage infection is a common route to escape silencing 10 . …”

Section: Silencing Of Chloramphenicol Resistance On Agar Platessupporting

confidence: 93%

“…The results of this work and earlier work 10 suggest that noninfected cells represent 1-10% of the final population, and are responsible for most of the nonsilenced phenotypes observed. A variety of routes to M13-resistance are known, with the most common being mutational loss of pilus expression 24 .…”

Section: Discussionsupporting

confidence: 62%

“…This protocol reproduces and expands on earlier work using phagemid-born sRNA cassettes 10 . First, a packaged phagemid is introduced to a batch culture of E. coli cells and used to silence expression of the fluorescent protein mKate2.…”

Section: Introductionmentioning

confidence: 62%

“…Note: For a fluorescent protein target, the silencing effect can be quantified directly by fluorometry 10 . Alternately, phenotypic assays may be used to observe the phenotypic consequences of gene knockdown 8 .…”

Section: Infection With Packaged Phagemids For Silencingmentioning

confidence: 99%

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Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

Bernheim

Libis

Lindner

et al. 2016

JoVE

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RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISCexpressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

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Section: Silencing Of Chloramphenicol Resistance On Agar Platessupporting

confidence: 93%

Section: Discussionsupporting

confidence: 62%

Section: Introductionmentioning

confidence: 62%

Section: Infection With Packaged Phagemids For Silencingmentioning

confidence: 99%

See 2 more Smart Citations

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

Bernheim

Libis

Lindner

et al. 2016

JoVE

Self Cite

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show abstract

“…Additionally, phagemid-induced expression of small regulatory RNAs has been used to knock down the expression of resistance genes in order to recover antibiotic susceptibility [52].…”

Section: Bacteriophage-based Therapeuticsmentioning

confidence: 99%