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LoxTnSeq: Transposon mutagenesis coupled with ultra-sequencing to study large random genome reductions
Link to bioRxiv paper:
http://biorxiv.org/cgi/content/short/2020.07.14.202044v1?rss=1
Authors: Shaw, D., Miravet-Verde, S., Pinero-Lambea, C., Serrano, L., Lluch-Senar, M.
Abstract:
Rational engineering in synthetic biology requires preliminary knowledge of which genomic regions are dispensable. Typically, these efforts are guided by transposon mutagenesis studies, coupled to ultra-sequencing (TnSeq) which determine single gene essentiality. However, epistatic interactions can rapidly alter these profiles post deletion, leading to the redundancy of these 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, with ultra-sequencing. When LoxTnSeq was applied to the genome reduced bacterium Mycoplasma pneumoniae, we obtained a mutant pool containing 285 unique deletions. These deletions spanned from >50 bp to 28Kb, which represents 21% of the total genome. LoxTnSeq also highlighted large regions of non-essential genes that could be removed simultaneously, and other similar regions that could not, providing a guide for future genome reductions.
Copy rights belong to original authors. Visit the link for more info
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Link to bioRxiv paper:
http://biorxiv.org/cgi/content/short/2020.07.14.202044v1?rss=1
Authors: Shaw, D., Miravet-Verde, S., Pinero-Lambea, C., Serrano, L., Lluch-Senar, M.
Abstract:
Rational engineering in synthetic biology requires preliminary knowledge of which genomic regions are dispensable. Typically, these efforts are guided by transposon mutagenesis studies, coupled to ultra-sequencing (TnSeq) which determine single gene essentiality. However, epistatic interactions can rapidly alter these profiles post deletion, leading to the redundancy of these 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, with ultra-sequencing. When LoxTnSeq was applied to the genome reduced bacterium Mycoplasma pneumoniae, we obtained a mutant pool containing 285 unique deletions. These deletions spanned from >50 bp to 28Kb, which represents 21% of the total genome. LoxTnSeq also highlighted large regions of non-essential genes that could be removed simultaneously, and other similar regions that could not, providing a guide for future genome reductions.
Copy rights belong to original authors. Visit the link for more info