ArticleApplied and environmental microbiology2010
New vector system for random, single-step integration of multiple copies of DNA into the Rhodococcus genome.
Article in Applied and environmental microbiology, 2010. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 5 papers.
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Who cites it
5 citing papers in PubMed.
- Advancing Desulfurization in the Model BiocatalystApplied and environmental microbiology · 2023Article
- Bioconversion of vitamin DBiotechnology for biofuels and bioproducts · 2022Review
- Single-Homology-Arm Linear DNA Recombination by the Nonhomologous End Joining Pathway as a Novel and Simple Gene Inactivation Method: a Proof-of-Concept Study inApplied and environmental microbiology · 2018Article
- Biodegradation of the organic disulfide 4,4'-dithiodibutyric acid by Rhodococcus spp.Applied and environmental microbiology · 2015Article
- A robust system for production of minicircle DNA vectors.Nature biotechnology · 2010Article
Corrections and comments
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Authors and funding
4 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
We designed a new vector system for creating a random mutant library with multiple integrations of DNA fragments into the Rhodococcus genome in a single step. For this, we cotransformed two vectors into Rhodococcus by electroporation: pTip-istAB-sacB regulates the expression of the transposase (IstA) and its helper protein (IstB) under the influence of a thiostrepton-inducible promoter, and pRTSK-sacB provides the transposable-marker DNA. Both are multicopy vectors that are stable in the host cells; transposition of the transposable-marker DNA occurs only after the induction of IstA/IstB expression. With the addition of thiostrepton, all cultured cells harboring the two vectors, irrespective of the volume, can be mutated by random insertion of the transposable-marker DNA into their genome. Among the generated mutants examined, 30% showed multiple (two to five) insertion copies. The multiple integrated DNA copies were stable in the genome for more than 80 generations of serial growth without the addition of any selective antibiotics. This system can also be used for integrating various copy numbers of stably maintained protein expression cassettes in the host cell genome to modulate the expression level of biologically active recombinant proteins. We successfully applied this system to integrate multiple copies of expression cassettes for proline iminopeptidase and vitamin D(3) hydroxylase into the Rhodococcus genome and verified that the clones containing double or multiple copies of the integrated cassettes produced higher levels and showed higher enzymatic activities of the target protein than clones with only a single copy of integration.
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Registered trials
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