Table 1 Chromosome engineering techniques widely used in E.coli

λ-Red recombination

The λ-Red recombination (or recombineering) approach has been successfully implemented in many studies to engineer specific sites in the E. coli chromosome [17]. In this approach, chromosomal sequences are replaced by a linear DNA fragment (flanked with sequences homologous to the region of interest) through the use of a temperature-sensitive plasmid that expresses either the Red recombinase genes (bet, gam and exo) from λ-phage [17] or the RecET proteins from Rac prophage [18] upon induction. The linear DNA fragment of interest is usually synthesized via PCR, in which case the homologous sequences (~50 bases) are introduced through the employed primers. The recombined strain is selected using a constitutively expressed antibiotic marker that is integrated into the chromosome along with the insert of interest. This technique may also be combined with FLP/FRT-based recombination, in which the antibiotic marker is flanked by FRT sites that allow it to be recombined out using the flippase (FLP) enzyme [19]. In this way, the created strain may be employed in multiple rounds of chromosome engineering using the same antibiotic marker [17, 20].

Generalized P1 phage transduction

Generalized P1 phage transduction is widely used to transfer mutations from one E. coli strain to another with the same genetic background [26, 27]. This approach is based on the fact that virulent P1 phages commit errors while packaging their DNA into coat proteins: instead of packaging their own genome, they package lysed host chromosomal DNA fragments [28–30]. Such mis-packaged phages form approximately ~5 % of the total phage population in a lysate. When they are transduced into a different host, the chromosome fragment may be inserted precisely at a homologous site using the RecA-dependent system [26]. Using this approach, multiple insertions can be made into the ~4.6 Mb chromosome of same E. coli strain, provided that they are separated by ~100 kb [31]. A combination of λ-Red recombination, FLP/FRT recombination, and P1 phage transduction methods can also be used to introduce multiple insertions into a single E. coli strain [6, 15].