Methylotroph cloning vehicle

Patent · Tue Apr 25 00:00:00 EDT 1989

OSTI ID:866931

Hanson, Richard S ^[1]; Allen, Larry N ^[2]

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1. Deephaven, MN
2. Excelsior, MN

A cloning vehicle comprising: a replication determinant effective for replicating the vehicle in a non-C.sub.1 -utilizing host and in a C.sub.1 -utilizing host; DNA effective to allow the vehicle to be mobilized from the non-C.sub.1 -utilizing host to the C.sub.1 -utilizing host; DNA providing resistance to two antibiotics to which the wild-type C.sub.1 -utilizing host is susceptible, each of the antibiotic resistance markers having a recognition site for a restriction endonuclease; a cos site; and a means for preventing replication in the C.sub.1 -utilizing host. The vehicle is used for complementation mapping as follows. DNA comprising a gene from the C.sub.1 -utilizing organism is inserted at the restriction nuclease recognition site, inactivating the antibiotic resistance marker at that site. The vehicle can then be used to form a cosmid structure to infect the non-C.sub.1 -utilizing (e.g., E. coli) host, and then conjugated with a selected C.sub.1 -utilizing mutant. Resistance to the other antibiotic by the mutant is a marker of the conjugation. Other phenotypical changes in the mutant, e.g., loss of an auxotrophic trait, is attributed to the C.sub.1 gene. The vector is also used to inactivate genes whose protein products catalyze side reactions that divert compounds from a biosynthetic pathway to a desired product, thereby producing an organism that makes the desired product in higher yields.

• Research Organization: Univ. of Minnesota, Minneapolis, MN (United States)
• DOE Contract Number: AC02-82ER12029
• Assignee: Regents of University of Minnesota (Minneapolis, MN)
• Patent Number(s): US 4824786
• Application Number: 07/107,244
• OSTI ID: 866931
• Country of Publication: United States
• Language: English

Related Subjects

methylotroph
cloning
vehicle
comprising
replication
determinant
effective
replicating
non-c
-utilizing
host
dna
allow
mobilized
providing
resistance
antibiotics
wild-type
susceptible
antibiotic
markers
recognition
site
restriction
endonuclease
cos
means
preventing
complementation
mapping
follows
organism
inserted
nuclease
inactivating
marker
form
cosmid
structure
infect
coli
conjugated
selected
mutant
conjugation
phenotypical
changes
loss
auxotrophic
trait
attributed
vector
inactivate
genes
protein
products
catalyze
reactions
divert
compounds
biosynthetic
pathway
desired
product
producing
makes
yields
-utilizing host
cloning vehicle
desired product
vehicle comprising
restriction endonuclease
nuclease recognition
/435/999/