Improved seamless mutagenesis by recombineering using ccdB for counterselection

Abstract

Recombineering, which is the use of homologous recombination for DNA engineering in Escherichia coli, usually uses antibiotic selection to identify the intended recombinant. When combined in a second step with counterselection using a small molecule toxin, seamless products can be obtained. Here, we report the advantages of a genetic strategy using CcdB as the counterselectable agent. Expression of CcdB is toxic to E. coli in the absence of the CcdA antidote so counterselection is initiated by the removal of CcdA expression. CcdB counterselection is robust and does not require titrations or experiment-to-experiment optimization. Because counterselection strategies necessarily differ according to the copy number of the target, we describe two variations. For multi-copy targets, we use two E. coli hosts so that counterselection is exerted by the transformation step that is needed to separate the recombined and unrecombined plasmids. For single copy targets, we put the ccdA gene onto the temperature-sensitive pSC101 Red expression plasmid so that counterselection is exerted by the standard temperature shift to remove the expression plasmid. To reduce unwanted intramolecular recombination, we also combined CcdB counterselection with Redα omission. These options improve the use of counterselection in recombineering with BACs, plasmids and the E. coli chromosome.

A CRISPR-Cas9 Assisted Non-Homologous End-Joining Strategy for One-step Engineering of Bacterial Genome

“…Various improved genome engineering methods based on phage recombination systems were developed; but while they facilitated the reprogramming of complex biological processes and accelerated the advance of metabolic engineering and synthetic biology, all of them required the construction of a specific homologous DNA editing template, usually also containing a selective marker, for each genomic locus1293031. This resulted in a labor-intensive and time-consuming procedure, especially for large-scale multiplex or iterative whole-genome engineering9 (Table 2).…”

A Novel Genome-Editing Platform for Drug-Resistant Acinetobacter baumannii Reveals an AdeR-Unrelated Tigecycline Resistance Mechanism

“…Several sucrose passages were required for successful plasmid removal, indicating the inefficiency of this counterselection cassette (17). SacB is also prone to accumulate inactivation mutations due to its slight cytotoxicity even in the absence of sucrose, making the use of this counterselection marker disadvantageous in A. baumannii (25).…”

“…In bacteria, expression of Tpm enzymatically detoxifies tellurite by a reduction and methylation of tellurite to produce volatile dimethyl telluride (24). The sacB gene, widely used as a counterselection cassette to initiate a second recombination event in different bacteria, was shown not to be capable of efficiently removing the integrated plasmid from the genome of A. baumannii and E. coli (17,25). During our design process we also noticed that SacB expression is slightly toxic even in the absence of sucrose, leading to the selection of inactivating mutations in the sacB gene.…”

Chemotaxis of Escherichia coli to Norepinephrine (NE) Requires Conversion of NE to 3,4-Dihydroxymandelic Acid

“…Selection for Ser ϩ transductants was accomplished by plating on minimal-glycerol TLHMB 1 agar, and the presence of the tsr mutations was screened by testing transductants on TB semisolid agar and picking isolates that did not form the outer (serine) chemotaxis ring. Glycerol was used as the carbon source in minimal medium because glucose adventitiously induces transcription of the ccdB (control of cell death) gene from the rhaB promoter in the kan insertion that allows counterselection for loss of the insertion in the presence of rhamnose (18). CcdB is a potent gyrase inhibitor (19) that is the toxin of an addiction system of the F plasmid of E. coli (20).…”