62 4 Rational Efforts to Streamline the Escherichia coli Genome
4.6.2.2 Linear DNA-Based Method
A more straightforward method applies a polymerase chain reaction (PCR)-
generated linear dsDNA fragment carrying flanking homology arms and genes
for selection and counterselection (Figure 4.3b). Recombination into the
genome is catalyzed by the lambda Red system [82], expressed typically from a
plasmid. The second recombination step, using a linear DNA fragment com-
posed of the flanking homology arms, is applied to replace the exogenous
sequences, creating a scarless deletion. Homology arms can be as short as
40 bp [83, 84] but work best when longer (1 kb; [47]). The method is straight-
forward, and even large deletions (~100 kb) can be obtained efficiently.
Incorporating a third homology box and a I-SceI cleavage site in the targeting
dsDNA fragment alleviates the need for recombination of a second targeting
fragment, accelerating the procedure. Induced I-SceI cleavage of the integrated
sequence stimulates intramolecular recombination between the third homol-
ogy box and a matching neighboring genomic sequence, resulting in a scarless
deletion [85](Figure 4.3b).4.6.2.3 Strategy for Piling Deletions
Accumulating deletions in a cell one by one is a labor-intensive endeavor. Some
simple strategical considerations help accelerating the process. Individual
deletion intermediates (e.g., unresolved co-integrates carrying a selection
marker) can be made and checked for fitness in a parallel fashion. Genomic
segments carrying the selected deletion intermediates can then be sequentially
transferred into the multiple deletion strain by cycles of P1 transduction andSequence to be deletedRecA-mediated
recombinationSequence to be deleted
A BAChromosome C BA BA BA AbR csm B AbRA AbR csm BElectroporation of
PCR fragmentElectroporation of
PCR fragmentA BAbRA B′ CA B′ C BA B′ C B^λ-Red-mediated integrationλ-Red-mediated
integration,
counterselection(b)I-SceI-induced DSBFigure 4.3 (Continued )