LoxTnSeq: random transposon insertions combined with cre/lox recombination and counterselection to generate large random genome reductions
Publication date
2021ISSN
1751-7915
Abstract
The removal of unwanted genetic material is a key aspect in many synthetic biology efforts and often requires preliminary knowledge of which genomic regions are dispensable. Typically, these efforts are guided by transposon mutagenesis studies, coupled to deepsequencing (TnSeq) to identify insertion points and gene essentiality. However, epistatic interactions can cause unforeseen changes in essentiality after the deletion of a gene, leading to the redundancy of these essentiality maps. Here, we present LoxTnSeq, a new methodology to generate and catalogue libraries of genome reduction mutants. LoxTnSeq combines random integration of lox sites by transposon mutagenesis, and the generation of mutants via Cre recombinase, catalogued via deep sequencing. When LoxTnSeq was applied to the naturally genome reduced bacterium Mycoplasma pneumoniae, we obtained a mutant pool containing 285 unique deletions. These deletions spanned from > 50 bp to 28 Kb, which represents 21% of the total genome. LoxTnSeq also highlighted large regions of non-essential genes that could be removed simultaneously, and other non-essential regions that could not, providing a guide for future genome reductions.
Document Type
Article
Document version
Published version
Language
English
Subject (CDU)
57 - Biological sciences in general
Keywords
Material genètic
Biologia sintètica
Genoma
Material genético
Biología sintética
Genoma
Genetic material
Synthetic biology
Genome
Pages
17
Publisher
John Wiley & Sons
Is part of
Microbial Biotechnology
Citation
Shaw, Daniel; Miravet-Verde, Samuel; Piñero-Lambea, Carlos [et al.]. LoxTnSeq: random transposon insertions combined with cre/lox recombination and counterselection to generate large random genome reductions. Microbial Biotechnology, 2020, p. 1-17. Disponible en: <https://sfamjournals.onlinelibrary.wiley.com/doi/full/10.1111/1751-7915.13714>. Fecha de acceso: 10 sep. 2021. DOI: 10.1111/1751-7915.13714
This item appears in the following Collection(s)
- Ciències Bàsiques [65]
Rights
2020 The Authors. Microbial Biotechnologypublished by Society for Applied Microbiology and John Wiley & Sons Ltd.This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permitsuse and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications oradaptations are made.bs_bs_banner
Except where otherwise noted, this item's license is described as https://creativecommons.org/licenses/by-nc-nd/4.0/