Method for isolation of insertion elements from coryneform bacteria

US5380657A · kind A · utility

Assignee

• Degussa AG · DE

Inventors

• Andreas Schaefer · Leverkusen, DE
• Anna-Hildegard Seep-Feldhaus · Bielefeld, DE
• Wolfgang Jaeger · Göppingen, DE
• Joern Kalinowski · Bielefeld, DE
• Wolfgang Wohlleben · Bielefeld, DE
• Alfred Puehler · Bielefeld, DE

Key dates

• Filing date: Mar 18, 1993
• Grant date: Jan 10, 1995
• Expiry date: Mar 18, 2013

Classification

• Technology area (CPC Y): Emerging Cross-Sectional Technologies
• CPC primary: Y10S435/843
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

A method 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: PA0 (1) The construction of a non-self-transferrable vector mobilizable from an E. coli mobilizer strain which vector is composed of PA1 (a) A DNA segment containing a replicon functional in E. coli, PA1 (b) A second DNA segment containing the DNA fragment coding for the mobilization function (Mob site containing the oriT), PA1 (c) A third DNA segment which recombines homologously in Gram-positive bacteria and/or contains a replicon functional in coryneform bacteria, PA1 (d) A DNA segment from Bacillus subtilis containing the sacB gens, PA0 (2) Transfer of this vector by means of conjugative transfer into the coryneform recipient strains, PA0 (3) Cultivation of the transconjugants containing the vector in an .about.10% sucrose-containing nutrient medium, PA0 (4) Lysis of the sucrose-resistant clones, cleaving of the plasmids with restriction endonucleases and analysis of the fragments.