Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants

✍ Scribed by Friedman, Alan M.; Long, Sharon R.; Brown, Susan E.; Buikema, William J.; Ausubel, Frederick M.

• Book ID: 121295004
• Publisher: Elsevier Science
• Year: 1982
• Tongue: English
• Weight: 664 KB
• Volume: 18
• Category: Article
• ISSN: 0378-1119
• DOI: 10.1016/0378-1119(82)90167-6

✦ Synopsis

We have constructed a cosmid derivative of the low copy-number broad host-range cloning vector pRK290 (Ditta et al., 1980) by inserting a 1.6kb Bg/II fragment containing X cos into the unique BglII site in pRK290. The new vector, pLAFR1, is 21.6 kb long, confers tetracycline resistance, contains a unique EcoRI site, and can be mobilized into and stably replicates within many Gram-negative hosts. We constructed a clone bank of Rhizobium meliloti DNA in pLAFR1 using a partial EcoRI digest. The mean insert size was 23.1 kb. When the clone bank was mated (en masse) from Escherichia coli to various R. meliloti auxotrophic mutants, tetracycline-resistant (Tc') transconjugants were obtained at frequencies ranging from 0.1 to 0.8, and among these, prototrophic colonies were obtained at frequencies ranging from 0.001 to 0.007. pLAFR1 cosmids were mobilized from R. meliloti prototrophic colonies into E. coli and then reintroduced into R. meliloti auxotrophs. In most cases, 100% of these latter Tc' transconjugants were prototrophic.