US 5,633,154 AGranted

Method for location of insertion elements

AssigneeDegussa AG

InventorsAndreas Schaefer, Anna-Hildegard Seep-Feldhaus, Wolfgang Jaeger, Joern Kalinowski, Wolfgang Wohlleben, Alfred Puehler

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Abstract

A method of locating insertion elements (IS elements) or transposons in coryneform bacteria, a positive selection system suitable for the above, the IS elements found in this manner and their use, is disclosed. The method involves: (1) The construction of a non-self-transferrable vector mobilizable from an E. coli mobilizer strain which vector is composed of (a) A DNA segment containing a replicon functional in E. coli, (b) A second DNA segment containing the DNA fragment coding for the mobilization function (Mob site containing the oriT), (c) A third DNA segment which recombines homologously in Gram-positive bacteria and/or contains a replicon functional in coryneform bacteria, (d) A DNA segment from Bacillus subtilis containing the sacB gene, (2) Transfer of this vector by means of conjugative transfer into the coryneform recipient strains, (3) Cultivation of the transconjugants containing the vector in an .about.10% sucrose-containing nutrient medium, (4) Lysis of the sucrose-resistant clones, cleaving of the plasmids with restriction endonucleases and analysis of the fragments.

Claims2

Dependent claims

A process of mutagenizing coryneform bacteria comprising

(a) constructing a non-self-transferrable vector which can be mobilized from an E. coli mobilizer strain, said vector comprising
(1) a DNA segment containing a replicon functional in E. coli,
(2) a second DNA segment containing the DNA fragment coding for the Mob site containing the oriT,
(3) a third DNA segment which recombines homologously in Gram-positive bacteria, or contains a replicon functional in coryneform bacteria, or recombines homologously in Gram-positive bacteria and contains a replicon functional in coryneform bacteria, and
(4) a DNA segment containing an IS element, wherein said insertion element is (i) ISCg1 from C. glutamicum, C. fascians, C. herculis and B. flavum, comprising approximately 1.45 kb and with inversely repetitive ends with a length of approximately 24 bp, (ii) ISBl1 from B. lactofermentum comprising approximately 1.45 kb and with inversely repetitive ends approximately 26 bp long, or (iii) ISRF1 from C. fascians, comprising approximately 1.3 kb and with inversely repetitive ends approximately 18 bp long,
(b) transferring said vector by means of conjugative transfer into a coryneform recipient strain to form transconjugants, and
(c) cultivating said transconjugants.

4 more elements

Dependencies are inferred from the claim text.

Facts

Application: 08/336,069Filed 4 Nov 1994 · division
Priority: DE 42 08 785.619 Mar 1992
Granted: 27 May 1997
Term ends: Not estimatedThe source records don’t support an estimate.

Classification11 codes

C12N 9/1055
Hexosyltransferases: levansucrase
C07K 14/34
From bacteria: from Corynebacterium
C12N 15/11
Recombinant DNA-technology: dNA or RNA fragments; Modified forms thereof ; Non-coding nucleic acids having a biological activity
C12N 15/77
Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora: for Corynebacterium;…