Lox’d in translation: Contradictions in the nomenclature surrounding common lox site mutants and their implications in experiments

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.

Mechanisms of Cre recombinase synaptic complex assembly and activation illuminated by Cryo-EM

“…Each loxP site comprises two palindromic 13-base pair (bp) recombinase binding elements (RBEs) that are recognized in antiparallel fashion by two Cre proteins (Figure 1A and B). The RBEs are separated by an 8-bp asymmetric spacer that determines the orientation of each site and the outcome of recombination reactions ( 9–12 ). Four molecules of Cre assemble with two loxP DNA sites, in alternating fashion, and tyrosine residues of each protomer perform a series of strand cleavages, exchanges and ligations, forming covalent 3′-phosphotyrosine and Holliday junction intermediates to generate recombinant products.…”

A toolbox for manipulating the genome of the major goat pathogen, Mycoplasma capricolum subsp. capripneumoniae

“…S1, available in the online version of this article) using the primer pairs MccpPeptS41pMT85gentPRS-F/MccpPeptS41pMT85gentPRS-F. The second one was produced with the primer pairs MccpPept-S41LOX66pMT85gentPRS-F/MccpPeptS41LOX71pMT85gentPRS-R in order to include loxP sites [55] in the amplicon (Fig. S2).…”

“…The first template was made of (i) a centromere (CEN6), an autonomously replicating sequence (ARSH4), and the HIS3 selection marker for selection in yeast, (ii) a gentamicin resistance marker for selection in mycoplasma cells, and (iii) 60 bp recombination arms placed at each extremity, identical to the sequences flanking the target gene. The second template was indistinguishable from the first except for the presence of two 34 bp loxP sites [55] flanking the Cre targets, i.e. the gentamicin resistance marker and the yeast components that must be removed to obtain seamless mutants (Fig.…”