Construction and characterization of F plasmid‐based expression vectors

Jones, Kristala L.; Keasling, Jay D.

doi:10.1002/(sici)1097-0290(19980920)59:6<659::aid-bit1>3.3.co;2-1

Cited by 1 publication

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“…Via these sites I-SceI was cloned into the pROCOLI vector downstream of the arabinose inducible pBAD promoter. This plasmid is a derivative of vector plkJ12 25 where the ampicillin resistance marker was changed to FRT-flanked chloramphenicol resistance gene. From the pROCOLI vector the pBAD-I-SceI fragment was amplified by PCR using primers BglII_SceI_sense and BglII_SceI_as.…”

Section: Methodsmentioning

confidence: 99%

Fast and antibiotic free genome integration into Escherichia coli chromosome

Egger

Tauer

Cserjan‐Puschmann

et al. 2020

Sci Rep

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Genome-based Escherichia coli expression systems are superior to conventional plasmid-based systems as the metabolic load triggered by recombinant compounds is significantly reduced. The efficiency of T7-based transcription compensates for low gene dosage (single copy) and facilitates high product formation rates. While common Gene Bridges’ λ-red mediated recombination technique for site directed integration of genes into the host genome is very efficient, selection for positive clones is based on antibiotic resistance markers and removal thereof is often time consuming. For the generation of industrial production strains, flexibility in terms of integration site is not required, yet time from gene design to a stable clone is a quite relevant parameter. In this study, we developed a fast, efficient and antibiotic-free integration method for E. coli as production strain. We combined the λ-red recombination system with the site-directed homing endonuclease I from Saccharaomyces cerevisiae (I-SceI) for selection. In a first step, λ-red proteins are performing genome integration of a linear, antibiotic marker-free integration cassette. The engineered host strain carries the I-SceI restriction sequence at the attTn7 site, where the integration event happens. After homologous recombination and integration at the target site, site-specific genome cleavage by endonuclease I-SceI is induced, thereby killing all cells still containing an intact I-SceI site. In case of positive recombination events, the genomic I-SceI site is deleted and cleavage is no longer possible. Since plasmids are designed to contain another I-SceI restriction site they are destroyed by self-cleavage, a procedure replacing the time-consuming plasmid curing. The new plasmid-based “All-In-One” genome integration method facilitates significantly accelerated generation of genome-integrated production strains in 4 steps.

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Section: Methodsmentioning

confidence: 99%