Abstract
The ability to conduct advanced functional genomic studies of the thousands of
38 sequenced bacteria has been hampered by the lack of available tools for making high39
throughput chromosomal manipulations in a systematic manner that can be applied across
40 diverse species. In this work, we highlight the use of synthetic biological tools to
41 assemble custom suicide vectors with reusable and interchangeable DNA “parts” to
42 facilitate chromosomal modification at designated loci. These constructs enable an array
43 of downstream applications including gene replacement and creation of gene fusions with
44 affinity purification or localization tags. We employed this approach to engineer
45 chromosomal modifications in a bacterium that has previously proven difficult to
46 manipulate genetically, Desulfovibrio vulgaris Hildenborough, to generate a library of
47 662 strains. Furthermore, we demonstrate how these modifications can be used for
48 examining metabolic pathways, protein-protein interactions, and protein localization. The
49 ubiquity of suicide constructs in gene replacement throughout biology suggests that this
50 approach can be applied to engineer a broad range of species for a diverse array of
51 systems biological applications and is amenable to high-throughput implementation.