Method for location of insertion elements

US5633154A · 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: Nov 4, 1994
• Grant date: May 27, 1997
• Expiry date: Nov 4, 2014

Classification

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

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: 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 gene, 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.