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DNA Res. 2016 Jun;23(3):263-70. doi: 10.1093/dnares/dsw015. Epub 2016 Apr 15.

All-in-one construct for genome engineering using Cre-lox technology.

Author information

1
Departament de Bioquímica i Biologia Molecular and Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Cerdanyola del Vallès 0819, Spain.
2
Departament de Bioquímica i Biologia Molecular and Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Cerdanyola del Vallès 0819, Spain jaume.pinyol@uab.cat.

Abstract

Mycoplasma genitalium is an appealing model of a minimal cell and synthetic biology study, and it was one of the first organisms whose genome was fully sequenced and chemically synthesized. Despite its usefulness as a model organism, many genetic tools well established for other microorganisms are not currently available in mycoplasmas. We have developed several vectors to adapt the Cre-lox technology for genome engineering in M. genitalium, providing an all-in-one construct that could be also useful to obtain unmarked genetic modifications in many other slow growing microorganisms. This construct contains a modified promoter sequence based in TetR system that exhibits an enhanced control on Cre recombinase expression, virtually abolishing the presence of this recombinase in the absence of inducer. This allows to introduce the Cre recombinase gene and the desired genetic modification in a single transformation step. In addition, this inducible promoter may be a very promising tool for a wide range of molecular applications.

KEYWORDS:

Cre-lox technology; TetR-TetO inducible promoters; genome engineering; mycoplasmas

PMID:
27084897
PMCID:
PMC4909314
DOI:
10.1093/dnares/dsw015
[Indexed for MEDLINE]
Free PMC Article

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