LoxTnSeq: random transposon insertions combined with cre/lox recombination and counterselection to generate large random genome reductions
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- dc.contributor.author Shaw, Daniel, 1993-
- dc.contributor.author Miravet Verde, Samuel, 1992-
- dc.contributor.author Piñero-Lambea, Carlos
- dc.contributor.author Serrano Pubull, Luis, 1982-
- dc.contributor.author Lluch-Senar, Maria 1982-
- dc.date.accessioned 2021-03-18T09:17:36Z
- dc.date.available 2021-03-18T09:17:36Z
- dc.date.issued 2021
- dc.description.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.
- dc.description.sponsorship This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement 634942 (MycoSynVac) and was also financed by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme, under grant agreement 670216 (MYCOCHASSIS). We also acknowledge support of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership, the Centro de Excelencia Severo Ochoa and the CERCA Programme/Generalitat de Catalunya
- dc.format.mimetype application/pdf
- dc.identifier.citation Shaw D, Miravet-Verde S, Piñero-Lambea C, Serrano L, Lluch-Senar, M. LoxTnSeq: random transposon insertions combined with cre/lox recombination and counterselection to generate large random genome reductions. Microb Biotechnol. 2021;14(6):2403-19. DOI: 10.1111/1751-7915.13714
- dc.identifier.doi http://dx.doi.org/10.1111/1751-7915.13714
- dc.identifier.issn 1751-7915
- dc.identifier.uri http://hdl.handle.net/10230/46840
- dc.language.iso eng
- dc.publisher Wiley Open Access
- dc.relation.ispartof Microbial Biotechnology. 2021;14(6):2403-19
- dc.relation.projectID info:eu-repo/grantAgreement/EC/H2020/634942
- dc.relation.projectID info:eu-repo/grantAgreement/EC/H2020/670216
- dc.rights © 2020 Daniel Shaw et al. Microbial Biotechnology published 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 permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made
- dc.rights.accessRights info:eu-repo/semantics/openAccess
- dc.rights.uri https://creativecommons.org/licenses/by-nc-nd/4.0/
- dc.subject.other Genètica
- dc.subject.other Genòmica
- dc.title LoxTnSeq: random transposon insertions combined with cre/lox recombination and counterselection to generate large random genome reductions
- dc.type info:eu-repo/semantics/article
- dc.type.version info:eu-repo/semantics/publishedVersion