Project Number: 1931-21000-017-07
Project Type: Reimbursable

Start Date: Sep 15, 2006
End Date: Sep 14, 2009

Objective:
Identify highly effective enhancer insulators to prevent interaction among promoters in the Transgene Removal and Containment System (TRECS), and bacterial transcriptional repression systems to repress basal level expression of Cre and Flp in E. coli and Agrobacterium. Incorporate identified enhancer insulators and transcriptional repression systems into existing TRECS to improve its performance and utilization.

Approach:
Several potential nuclear matrix attachment regions (MARs) and putative enhancer insulators (EI) characterized in plants factors will be cloned and inserted between the 35S and AGIP promoters in 35S/AGIP::GUS construct, where the adjacent 35S promoter activates the flower-specific AGIP::GUS in vegetative tissue. Their ability to insulate the AGIP promoter from interference by the 35S promoter will be evaluated through analyzing GUS expression in leaf, root, and flower tissues through both histochemical staining and RT-PCR. Those that are able to prevent the AGIP::GUS vegetative expression will be identified as candidate EIs. A tetracycline repressor (tetR) that represses tetA expression by binding tet operators (tetO) in the tetA promoter will be first analyzed. Three copies of the tetO will be added to the 3¿ end of the AGIP to create an AGIP-tetO promoter that is then placed in front of Cre or Flp. The entire AGIP-tetO::Cre and AGIP-tetO::Flp fragments will be respectively inserted into A-TRECS-tetR vector, where the tetR (constitutively expressed in bacteria) is inserted to the outside of a T-DNA border in the TRECS vector. TetR molecules are expected to bind tetO in the AGIP-tetO promoter and completely repress the basal level expression of Cre or Flp, stabilizing TRECS constructs in bacteria. The identified EIs and repression systems will be incorporated into the existing TRECS to build a new generation of the TRECS platform which will be used for analyzing and comparing excision efficiency among different scissors genes, optimizing and engineering the identified scissors¿ efficiency.