Patent ID: 9109012
Filing Date: 2015-08-18
Classification: A61K,C07K,C12N

Abstract:
1. A method for selection and propagation of an antibiotic marker-free covalently closed, super-coiled DNA replicon in bacterial cells, comprising the steps of: a. cloning a RNA-OUT antisense gene into said DNA replicon, wherein a portion of the RNA-OUT gene sequence is complementary to an RNA-IN gene sequence and an ATG initiation site; b. transforming parent bacterial cells with the RNA-OUT antisense gene containing DNA replicon, said parent bacterial cells constitutively expressing a levansucrase gene under control of a RNA-IN sense RNA, so as to create transformed bacterial cells wherein said RNA-OUT gene is expressed and RNA-OUT antisense RNA is produced, said levansucrase gene comprising an ATG initiation site and linked to a heterologous constitutive RNA-IN promoter and an RNA-IN leader sequence, said RNA-IN sequence comprising a ribosomal binding site complementary to a portion of said RNA-OUT antisense gene, said RNA-IN promoter and said RNA-IN leader sequence comprising annealed primer pairs SEQ ID NO: 9 and SEQ ID NO: 10 wherein said expressed RNA-OUT antisense RNA hybridizes to said portion of said RNA-IN sequence and said ATG initiation site, thereby reducing the translation of said levansucrase gene such that the growth of said parent bacterial cells is inhibited in the presence of sucrose; c. isolating said transformed bacterial cells under selection by demonstrating growth in the presence of sucrose, and d. propagating said transformed bacterial cells under conditions that accumulate said DNA replicon.