title: Generalized schemes for high-throughput manipulation of the Desulfovibrio vulgaris genome.
abstract: The ability to conduct advanced functional genomic studies of the thousands of sequenced bacteria has been hampered by the lack of available tools for making high-throughput chromosomal manipulations in a systematic manner that can be applied across diverse species. In this work, we highlight the use of synthetic biological tools to assemble custom suicide vectors with reusable and interchangeable DNA "parts" to facilitate chromosomal modification at designated loci. These constructs enable an array of downstream applications, including gene replacement and the creation of gene fusions with affinity purification or localization tags. We employed this approach to engineer chromosomal modifications in a bacterium that has previously proven difficult to manipulate genetically, Desulfovibrio vulgaris Hildenborough, to generate a library of over 700 strains. Furthermore, we demonstrate how these modifications can be used for examining metabolic pathways, protein-protein interactions, and protein localization. The ubiquity of suicide constructs in gene replacement throughout biology suggests that this approach can be applied to engineer a broad range of species for a diverse array of systems biological applications and is amenable to high-throughput implementation.
authors: Physical Biosciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Mail Stop 978R412, Berkeley, CA 94720, USA. srchhabra@lbl.gov
authors: S R
authors: SR
authors: Chhabra
authors: G
authors: G
authors: Butland
authors: D A
authors: DA
authors: Elias
authors: J-M
authors: JM
authors: Chandonia
authors: O-Y
authors: OY
authors: Fok
authors: T R
authors: TR
authors: Juba
authors: A
authors: A
authors: Gorur
authors: S
authors: S
authors: Allen
authors: C M
authors: CM
authors: Leung
authors: K L
authors: KL
authors: Keller
authors: S
authors: S
authors: Reveco
authors: G M
authors: GM
authors: Zane
authors: E
authors: E
authors: Semkiw
authors: R
authors: R
authors: Prathapam
authors: B
authors: B
authors: Gold
authors: M
authors: M
authors: Singer
authors: M
authors: M
authors: Ouellet
authors: E D
authors: ED
authors: Szakal
authors: D
authors: D
authors: Jorgens
authors: M N
authors: MN
authors: Price
authors: H E
authors: HE
authors: Witkowska
authors: H R
authors: HR
authors: Beller
authors: A P
authors: AP
authors: Arkin
authors: T C
authors: TC
authors: Hazen
authors: M D
authors: MD
authors: Biggin
authors: M
authors: M
authors: Auer
authors: J D
authors: JD
authors: Wall
authors: J D
authors: JD
authors: Keasling
mesh_terms: D019677:Artificial Gene Fusion; D004269:DNA, Bacterial / Q000235:genetics*; D016969:Desulfovibrio vulgaris / Q000235:genetics*; D017353:Gene Deletion; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods*; D016680:Genome, Bacterial; D023281:Genomics / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D016254:Mutagenesis, Insertional / Q000379:methods; D011995:Recombination, Genetic
chemical_list: D004269:DNA, Bacterial