Synthetic Biology Parts, Devices and Applications

4.6 Targeted Deletion Techniiues 61

Selection of cointegrates

Sequence to

be deleted

Cloning

Electroporation

AbR

Plasmid integration

mediated by RecA

Recombinant PCR

Genome

AA

AA

A

A B

B

BB

B B

A B

AbR

Ori

AbR

Ori

A B

(a) Wild type Deletion mutant

Cleavage by I-SceI

Resolution by intramolecular recombination

I-SceI

I-SceI

I-SceI

Figure 4.3 General scheme of standard deletion procedures. (a) Overview of the circular
DNA-based method. Boxes A and B represent >500-bp DNA segments flanking the genomic
region to be deleted. AbR stands for an antibiotic resistance marker gene; ori indicates a
replication origin functioning only under permissive conditions. (b) Overview of the
λ-Red-mediated, linear DNA-based deletion method. Two alternative routes for generating
deletions are shown. A, B, and C represent arbitrarily chosen 40–60-bp DNA segments
(homology boxes). Arrowheads represent I-SceI cleavage sites. AbR and csm stand for an
antibiotic resistance marker and a counterselectable gene, respectively.