Conditional-Replication, Integration, Excision, and Retrieval Plasmid-Host Systems for Gene Structure-Function Studies of Bacteria

• DOI: 10.1128/jb.183.21.6384-6393.2001
• Publication Date: 2002-07-27T10:01:09Z
• Authors: Andreas Haldimann; Barry L. Wanner
• Keywords: T-DNA Binary system

ABSTRACT We have developed a series of powerful and versatile conditional-replication, integration, modular (CRIM) plasmids. CRIM plasmids can be replicated at medium or high copy numbers in different hosts for making gene (or mutant) libraries. They integrated single copies into the chromosomes Escherichia coli related bacteria to study function under normal physiological conditions. excised from chromosome, e.g., verify that phenotypes are caused by their presence. Furthermore, they retrieved singly en masse subsequent molecular analyses. chromosome site-specific recombination one five phage attachment sites. Integrants selected as antibiotic-resistant transformations. Since encode forms resistance, several used together same cell stable expression complex metabolic regulatory pathways diverse sources. Following integrants stably maintained absence antibiotic selection. Each plasmid has polylinker promoters ectopic inserted DNA. Their design allows easy construction new variants with combinations features. also report easily curable, low-copy-number helper encoding all requisite Int proteins alone respective Xis protein. These facilitate excision (“curing”), retrieval