Abstract
BackgroundCircular plasmid-mediated homologous recombination is commonly used for marker-less allelic replacement, exploiting the endogenous recombination machinery of the host. Common limitations of existing methods include high false positive rates due to mutations in counter-selection genes, and limited applicability to specific strains or growth media. Finally, solutions compatible with physical standards, such as the BioBrick™, are not currently available, although they proved to be successful in the design of other replicative or integrative plasmids.FindingsWe illustrate pBBknock, a novel BioBrick™-compatible vector for allelic replacement in Escherichia coli. It includes a temperature-sensitive replication origin and enables marker-less genome engineering via two homologous recombination events. Chloramphenicol resistance allows positive selection of clones after the first event, whereas a colorimetric assay based on the xylE gene provides a simple way to screen clones in which the second recombination event occurs. Here we successfully use pBBknock to delete the lactate dehydrogenase gene in E. coli W, a popular host used in metabolic engineering.ConclusionsCompared with other plasmid-based solutions, pBBknock has a broader application range, not being limited to specific strains or media. We expect that pBBknock will represent a versatile solution both for practitioners, also among the iGEM competition teams, and for research laboratories that use BioBrick™-based assembly procedures.Electronic supplementary materialThe online version of this article (doi:10.1186/s12575-016-0036-z) contains supplementary material, which is available to authorized users.
Highlights
Circular plasmid-mediated homologous recombination is commonly used for marker-less allelic replacement, exploiting the endogenous recombination machinery of the host
The I-SceI counter-selection system has been proposed to overcome such issues [7], but false positive clones due to mutations can still occur at high frequency [8]. This is a common feature of synthetic kill switches implemented via toxic genes [9], combination of multiple counter-selection systems has been reported to decrease the false positive rate [6]
This strategy, coupled with a lacZ gene-mediated blue/white screening, is successfully used in Gram positive bacteria [11], its use in E. coli would be limited to specific lacZ-mutant strains
Summary
Circular plasmid-mediated homologous recombination is commonly used for marker-less allelic replacement, exploiting the endogenous recombination machinery of the host. In such method, a mutated version of the target locus is cloned in a conditional-replication plasmid, together with the two DNA sequences flanking it. Methods have been proposed that use temperature-sensitive vectors without toxic genes, exploiting the integrated replication origin to stimulate the second recombination event in permissive (replicative) conditions [10]. This strategy, coupled with a lacZ gene-mediated blue/white screening, is successfully used in Gram positive bacteria [11], its use in E. coli would be limited to specific lacZ-mutant strains
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
