OPTIMIZING CELL SURFACE DISPLAY IN E COLI

DESCRIPTION: Surface display of foreign proteins on the outer membrane of bacterial cells provides a unique and potentially powerful means to screen for novel functionality. However, practical constraints on the implementation of this technology prevents its widespread application. One of the best characterized anchor proteins for display is a fusion between a fragment of the major E. coli lipoprotein (Lpp) and a fragment of the outer membrane protein, OmpA. Dr. Coleman proposes to use a fusion of Lpp-OmpA with the Green Fluorescent Protein (Lpp-OmpA-GFP) to help engineer a membrane anchor protein that is more stable in E. coli, has the optimum level of surface expression, allows multiple rounds of cell sorting, and which permits the bacteria to be grown to a higher cell density and at a higher temperature. An improved cell surface display system will allow a greater variety of proteins to be expressed under more manageable conditions, providing an impetus for the advancement of such diverse technologies as antibody development and enzyme evolution. $ = TOTAL AWARD AMTS & NOT LIMITED TO PORTION OF PROJECT RELATED TO SUBJECT OF SEARCH SUBPROJECT $ = TOTAL AWARD AMOUNT DIVIDED BY NUMBER OF SUBPROJECTS SOURCE: CRISP FORMAT F FY 97 LAST UPDATE 04-07-98 1QUERY 1536 ID SEARCH 06/01/98 PAGE 98 --PROJECT NUMBER......1 R43 AI42481-01 INVESTIGATOR NAME/ADDRESS FY 97 THOMAS, DAVID C IRG/INTRAMURAL UNIT..ZRG2 ONCOR INC AWARD AMOUNT......... $99,500 209 PERRY PARKWAY GAITHERSBURG, MD 20877 PERFORMING ORGANIZATION: ONCOR, INC. TITLE CASCADE ROLLING CIRCLE AMPLIFICATION FOR DNA DIAGNOSTICS ABSTRACT: Many current nucleic acid amplification systems for detecting pathogenic agents may not yield consistent results because optimal amplification conditions vary with each target sequence. Cascade rolling circle amplification (CRCA) is a novel isothermal amplification system that uses generic primers to amplify circularized probes instead of the target. It is rapid, highly sensitive, and easily multiplexed, consistently yielding over a billion-fold amplification within one hour without using a thermocycler. If one of the primers is an energy transfer-labeled primer, homogeneous fluorescence detection of the CRCA product is possible within a closed system. The speed and ease of the assay make this technology suitable for field work or use in developing countries, as well as for simpler automation in diagnostic applications. The feasibility of this system has been demonstrated in preliminary experiments. Phase I objectives include optimization of ligation and amplification conditions to achieve maximum signal strength and sensitivity, and optimization of energy transfer-labeled primer structures that yield the highest fluorescence signal to background ratio without compromising amplification efficiency. The speed and sensitivity of the proposed method will be compared with traditional techniques using a pathogenic target Phase II studies will focus on clinical applications of this system.