LoxTnSeq: random transposon insertions combined with cre/lox recombination and counterselection to generate large random genome reductions
| Autor: | Luis Serrano, Maria Lluch-Senar, Daniel Shaw, Samuel Miravet-Verde, Carlos Pinero |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Transposable element
Mutant Material genético Cre recombinase Bioengineering Computational biology Biology Applied Microbiology and Biotechnology Biochemistry Genome Deep sequencing 03 medical and health sciences Synthetic biology Material genètic Genetic material Gene Research Articles Genoma 030304 developmental biology Recombination Genetic Biologia sintètica 0303 health sciences Integrases 030306 microbiology Genomics Mutagenesis Insertional Genòmica DNA Transposable Elements Transposon mutagenesis Cre-Lox recombination Biología sintética Genètica TP248.13-248.65 Research Article Biotechnology |
| Zdroj: | Microbial Biotechnology, 14 (6) Microbial Biotechnology, Vol 14, Iss 6, Pp 2403-2419 (2021) Microbial Biotechnology |
| ISSN: | 1751-7915 1751-7907 |
| Popis: | 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. Microbial Biotechnology, 14 (6) ISSN:1751-7915 ISSN:1751-7907 |
| Databáze: | OpenAIRE |
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