Patent ID: 6048694
Filing Date: 2000-04-11
Classification: C12N

Abstract:
A method for the positive selection of Gram positive bacterial host cells transformed with heterologus DNA comprising:(i) constructing a positive selection vector comprising:(a) a levansucrase gene encoding an active levansucrase polymerase said gene having a signal peptide processing mutation causing a reduction in signal peptide processing, the gene further comprising compatible restriction sites useful for the insertion of heterologous DNA;(b) a gene encoding antibiotic resistance;(c) a host specific origin of replication for maintaining the vector as multicopy; and(d) suitable regulatory sequences for the regulation and expression of the levansucrase gene;(ii) cloning heterologous DNA into said compatible restriction sites of said levansucrase gene wherein the levansucrase polymerase activity is destroyed;(iii) transforming a competent Gram positive host cell with the vector of step (ii) wherein said host cell does not possess the ability to metabolize sucrose;(iv) incubating said transformed host cells in the presence of a suitable growth medium containing sucrose; and(v) isolating transformants which grow in the presence of sucrose.