C - Chemistry – Metallurgy – 12 – N
Patent
C - Chemistry, Metallurgy
12
N
195/1.14, 195/1.
C12N 15/74 (2006.01) C12N 15/11 (2006.01) C12N 15/73 (2006.01)
Patent
CA 1325190
ABSTRACT OF THE DISCLOSURE Several embodiments of DNA cassettes and plasmids and cosmids containing portable DNA cassettes are provided herein. One embodiment is a portable DNA cassette into which a mob region of a conjugative, mobilizable, wide-host range plasmid has been cloned, the portable DNA cassette having endonuclease restriction sites at both termini for subsequent cutting by a selected restriction endonuclease. Another embodiment is a plasmid containing a portable DNA cassette into which a mob region of a conjugative, mobilizable, wide-host range plasmid has been cloned, and having endonuclease restriction sites at both termini for subsequent cutting by a selected restriction endonuclease. A third embodiment is a cosmid containing a portable DNA cassette, the portable DNA cassette being one into which a mob region of a conjugative, mobilizable wide- host plasmid has been cloned, and one into which a minicos region from bacteriophage lambda has been cloned, and also having selected endonuclease restriction sites at both termini for subsequent cutting by a selected restriction endonuclease. Each of these cosmids can be packaged with the lysate of a lambda or lambdoid phage. The packaged product may be transduced into Escherichia coli. Those hybrid bacteria can then be transferred to any gram-negative bacteria, e.g. to Rhizobium Spp or Alcaligenes eutrophus. The invention also provided processes for the construction of a portable DNA cassette, a process for the construction of a plasmid ABSTRACT OF THE DISCLOSURE (page 2) containing a portable DNA cassette, for the construction of a cosmid containing a portable DNA cassette, for packaging a cosmid containing a portable DNA cassette into bacteriophage lambda, and for mobilizing a cosmid containing a portable DNA cassette. The yield of clones containing these hybrids, particularly for those of the larger size class, is several hundred or thousand fold better than that obtained through normal transformation. Furthermore, by the use of small plasmids, which themselves are not packaged efficiently, the background of non-hybrid clones is effectively reduced in a single step without resort to the elaborate selection or screening procedures normally used in cloning procedures.
461600
Iyer Venkatran N.
Selvaraj Gopalan
Iyer Venkatran N.
Marcus & Associates
Selvaraj Gopalan
LandOfFree
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