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bioRxiv · 10.1101/2020.07.14.202044

LoxTnSeq: Transposon mutagenesis coupled with ultra-sequencing to study large random genome reductions

Shaw, D.·Miravet-Verde, S.·Pinero-Lambea, C.·Serrano, L.·Lluch-Senar, M.
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Abstract

The Cre-Lox system is a highly versatile and powerful DNA recombinase mechanism, mainly used in genetic engineering to insert or remove desired DNA sequences. It is widely utilised across multiple fields of biology, with applications ranging from plants, to mammals, to microbes. A key feature of this system is its ability to allow recombination between mutant lox sites, traditionally named lox66 and lox71, to create a functionally inactive double mutant lox72 site. However, a large portion of the published literature has incorrectly annotated these mutant lox sites, which in turn can lead to difficulties in replication of methods, design of proper vectors, and confusion over the proper nomenclature. Here, we demonstrate common errors in annotations, the impacts they can have on experimental viability, and a standardised naming convention. We also show an example of how this incorrect annotation can induce toxic effects in bacteria that lack optimal DNA repair systems, exemplified by Mycoplasma pneumoniae. Data SummaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

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Shaw, D., Miravet-Verde, S., Pinero-Lambea, C., Serrano, L., Lluch-Senar, M.. 2020-07-14. LoxTnSeq: Transposon mutagenesis coupled with ultra-sequencing to study large random genome reductions. https://doi.org/10.1101/2020.07.14.202044

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