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Citation
Tags
HERO ID
1677533
Reference Type
Journal Article
Title
Knock-in/Knock-out (KIKO) vectors for rapid integration of large DNA sequences, including whole metabolic pathways, onto the Escherichia coli chromosome at well-characterised loci
Author(s)
Sabri, S; Steen, JA; Bongers, M; Nielsen, LK; Vickers, CE
Year
2013
Is Peer Reviewed?
1
Journal
Microbial Cell Factories
ISSN:
1475-2859
EISSN:
14752859
Volume
12
Issue
1
Page Numbers
60
Language
English
PMID
23799955
DOI
10.1186/1475-2859-12-60
Web of Science Id
WOS:000321516100001
URL
http://microbialcellfactories.biomedcentral.com/articles/10.1186/1475-2859-12-60
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Abstract
BACKGROUND:
Metabolic engineering projects often require integration of multiple genes in order to control the desired phenotype. However, this often requires iterative rounds of engineering because many current insertion approaches are limited by the size of the DNA that can be transferred onto the chromosome. Consequently, construction of highly engineered strains is very time-consuming. A lack of well-characterised insertion loci is also problematic.
RESULTS:
A series of knock-in/knock-out (KIKO) vectors was constructed for integration of large DNA sequences onto the E. coli chromosome at well-defined loci. The KIKO plasmids target three nonessential genes/operons as insertion sites: arsB (an arsenite transporter); lacZ (beta-galactosidase); and rbsA-rbsR (a ribose metabolism operon). Two homologous 'arms' target each insertion locus; insertion is mediated by lamda Red recombinase through these arms. Between the arms is a multiple cloning site for the introduction of exogenous sequences and an antibiotic resistance marker (either chloramphenicol or kanamycin) for selection of positive recombinants. The resistance marker can subsequently be removed by flippase-mediated recombination. The insertion cassette is flanked by hairpin loops to isolate it from the effects of external transcription at the integration locus. To characterize each target locus, a xylanase reporter gene (xynA) was integrated onto the chromosomes of E. coli strains W and K-12 using the KIKO vectors. Expression levels varied between loci, with the arsB locus consistently showing the highest level of expression. To demonstrate the simultaneous use of all three loci in one strain, xynA, green fluorescent protein (gfp) and a sucrose catabolic operon (cscAKB) were introduced into lacZ, arsB and rbsAR respectively, and shown to be functional.
CONCLUSIONS:
The KIKO plasmids are a useful tool for efficient integration of large DNA fragments (including multiple genes and pathways) into E. coli. Chromosomal insertion provides stable expression without the need for continuous antibiotic selection. Three non-essential loci have been characterised as insertion loci; combinatorial insertion at all three loci can be performed in one strain. The largest insertion at a single site described here was 5.4 kb; we have used this method in other studies to insert a total of 7.3 kb at one locus and 11.3 kb across two loci. These vectors are particularly useful for integration of multigene cassettes for metabolic engineering applications.
Keywords
Chromosomal integration; Homologous recombination; Plasmid; Recombineering; E. coli; Xylanase; GFP; csc genes
Tags
IRIS
•
Arsenic Hazard ID
PubMed
Considered New
WOS
Considered New
2. Lit Search Updates through Oct 2015
PubMed
WOS
Considered
7. Other Studies through Oct 2015
Other
•
Arsenic (Inorganic)
1. Literature
Lit search updates through Oct 2015
3. Hazard ID Screening
Other potentially supporting studies
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