LoxTnSeq: random transposon insertions combined with cre/lox recombination and counterselection to generate large random genome reductions

Summary 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 i...

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Autores principales:	Daniel Shaw, Samuel Miravet‐Verde, Carlos Piñero‐Lambea, Luis Serrano, Maria Lluch‐Senar
Formato:	article
Lenguaje:	EN
Publicado:	Wiley 2021
Materias:	Biotechnology TP248.13-248.65
Acceso en línea:	https://doaj.org/article/37be770e951f4fe5b809c00854c92a1a
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spelling	oai:doaj.org-article:37be770e951f4fe5b809c00854c92a1a2021-11-18T15:39:52ZLoxTnSeq: random transposon insertions combined with cre/lox recombination and counterselection to generate large random genome reductions1751-791510.1111/1751-7915.13714https://doaj.org/article/37be770e951f4fe5b809c00854c92a1a2021-11-01T00:00:00Zhttps://doi.org/10.1111/1751-7915.13714https://doaj.org/toc/1751-7915Summary 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.Daniel ShawSamuel Miravet‐VerdeCarlos Piñero‐LambeaLuis SerranoMaria Lluch‐SenarWileyarticleBiotechnologyTP248.13-248.65ENMicrobial Biotechnology, Vol 14, Iss 6, Pp 2403-2419 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Biotechnology TP248.13-248.65
spellingShingle	Biotechnology TP248.13-248.65 Daniel Shaw Samuel Miravet‐Verde Carlos Piñero‐Lambea Luis Serrano Maria Lluch‐Senar LoxTnSeq: random transposon insertions combined with cre/lox recombination and counterselection to generate large random genome reductions
description	Summary 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.
format	article
author	Daniel Shaw Samuel Miravet‐Verde Carlos Piñero‐Lambea Luis Serrano Maria Lluch‐Senar
author_facet	Daniel Shaw Samuel Miravet‐Verde Carlos Piñero‐Lambea Luis Serrano Maria Lluch‐Senar
author_sort	Daniel Shaw
title	LoxTnSeq: random transposon insertions combined with cre/lox recombination and counterselection to generate large random genome reductions
title_short	LoxTnSeq: random transposon insertions combined with cre/lox recombination and counterselection to generate large random genome reductions
title_full	LoxTnSeq: random transposon insertions combined with cre/lox recombination and counterselection to generate large random genome reductions
title_fullStr	LoxTnSeq: random transposon insertions combined with cre/lox recombination and counterselection to generate large random genome reductions
title_full_unstemmed	LoxTnSeq: random transposon insertions combined with cre/lox recombination and counterselection to generate large random genome reductions
title_sort	loxtnseq: random transposon insertions combined with cre/lox recombination and counterselection to generate large random genome reductions
publisher	Wiley
publishDate	2021
url	https://doaj.org/article/37be770e951f4fe5b809c00854c92a1a
work_keys_str_mv	AT danielshaw loxtnseqrandomtransposoninsertionscombinedwithcreloxrecombinationandcounterselectiontogeneratelargerandomgenomereductions AT samuelmiravetverde loxtnseqrandomtransposoninsertionscombinedwithcreloxrecombinationandcounterselectiontogeneratelargerandomgenomereductions AT carlospinerolambea loxtnseqrandomtransposoninsertionscombinedwithcreloxrecombinationandcounterselectiontogeneratelargerandomgenomereductions AT luisserrano loxtnseqrandomtransposoninsertionscombinedwithcreloxrecombinationandcounterselectiontogeneratelargerandomgenomereductions AT marialluchsenar loxtnseqrandomtransposoninsertionscombinedwithcreloxrecombinationandcounterselectiontogeneratelargerandomgenomereductions
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LoxTnSeq: random transposon insertions combined with cre/lox recombination and counterselection to generate large random genome reductions

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