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Utilizing in vitro DNA assembly to engineer a synthetic T7 Nanoluc reporter phage for Escherichia coli detection.

08:00 EDT 30th March 2019 | BioPortfolio

Summary of "Utilizing in vitro DNA assembly to engineer a synthetic T7 Nanoluc reporter phage for Escherichia coli detection."

Bacteria have major role in regulating human health and disease, therefore, there is a continuing need to develop new detection methods and therapeutics to combat them. Bacteriophages can be used to infect specific bacteria, which make them good candidates for detecting and editing bacterial populations. However, creating phage-based detection assays is somewhat limited by the difficulties in the engineering of phages. We present here a synthetic biology strategy to engineer phages using a simple in vitro method. We used this method to insert a NanoLuc luciferase expression cassette into the T7 phage, in order to construct the NRGp6 reporter phage. The synthetic NRGp6 phage was used to efficiently detect low concentrations of Escherichia coli from liquid culture. We envision that our approach will benefit synthetic biologists for constructing different kinds of engineered phages, and enable new approaches for phage-based therapeutics and diagnostics.

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Name: Integrative biology : quantitative biosciences from nano to macro
ISSN: 1757-9708
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Medical and Biotech [MESH] Definitions

Bacteriophages whose genetic material is RNA, which is single-stranded in all except the Pseudomonas phage phi 6 (BACTERIOPHAGE PHI 6). All RNA phages infect their host bacteria via the host's surface pili. Some frequently encountered RNA phages are: BF23, F2, R17, fr, PhiCb5, PhiCb12r, PhiCb8r, PhiCb23r, 7s, PP7, Q beta phage, MS2 phage, and BACTERIOPHAGE PHI 6.

Biologically functional sequences of DNA chemically synthesized in vitro.

Specific loci on both the bacterial DNA (attB) and the phage DNA (attP) which delineate the sites where recombination takes place between them, as the phage DNA becomes integrated (inserted) into the BACTERIAL DNA during LYSOGENY.

The phenomenon by which a temperate phage incorporates itself into the DNA of a bacterial host, establishing a kind of symbiotic relation between PROPHAGE and bacterium which results in the perpetuation of the prophage in all the descendants of the bacterium. Upon induction (VIRUS ACTIVATION) by various agents, such as ultraviolet radiation, the phage is released, which then becomes virulent and lyses the bacterium.

Synthetic transcripts of a specific DNA molecule or fragment, made by an in vitro transcription system. This cRNA can be labeled with radioactive uracil and then used as a probe. (King & Stansfield, A Dictionary of Genetics, 4th ed)

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