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New vector system for random, single-step integration of multiple copies of DNA into the Rhodococcus genome.

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.

SUBMITTER: Sallam KI

PROVIDER: S-EPMC2849190 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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New vector system for random, single-step integration of multiple copies of DNA into the Rhodococcus genome.

Sallam Khalid Ibrahim KI Tamura Noriko N Imoto Noriko N Tamura Tomohiro T

Applied and environmental microbiology 20100212 8

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 ce ...[more]

PMID: 20154109

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Project description:Quantitative susceptibility mapping (QSM) was developed to estimate the spatial distribution of magnetic susceptibility from MR signal phase acquired using a gradient echo (GRE) sequence. The field-to-susceptibility inversion in QSM is known to be ill-posed and needs numerical stabilization through either regularization or data oversampling. The calculation of susceptibility through the multiple orientation sampling (COSMOS) method uses phase data acquired at three or more head orientations to achieve a well-conditioned field-to-susceptibility inversion and is often considered the gold standard for in vivo QSM. However, the conventional COSMOS approach, here named multistep COSMOS (MSCOSMOS), solves the dipole inversion from the local field derived from raw GRE phase through multiple steps of phase preprocessing. Error propagations between these consecutive phase processing steps can thus affect the final susceptibility quantification. On the other hand, recently proposed single-step QSM (SSQSM) methods aim to solve an integrated inversion from unprocessed or total phase to mitigate such error propagations but have been limited to single orientation QSM. This study therefore aimed to test the feasibility of using single-step COSMOS (SSCOSMOS) to jointly perform background field removal and dipole inversion with multiple orientation sampling, which could serve as a better standard for gauging SSQSM methods. We incorporated multiple spherical mean value (SMV) kernels of various radii with the dipole inversion in SSCOSMOS. QSM reconstructions with SSCOSMOS and MSCOSMOS were compared using both simulations with a numerical head phantom and in vivo human brain data. SSCOSMOS permitted integrated background removal and dipole inversion without the need to adjust any regularization parameters. In addition, with sufficiently large SMV kernels, SSCOSMOS performed consistently better than MSCOSMOS in all the tested error metrics in our simulations, giving better susceptibility quantification and smaller reconstruction error. Consistent tissue susceptibility values were obtained between SSCOSMOS and MSCOSMOS.

Dataset Information

New vector system for random, single-step integration of multiple copies of DNA into the Rhodococcus genome.

Publications

New vector system for random, single-step integration of multiple copies of DNA into the Rhodococcus genome.

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