We aim to design an E. coli-based detection system that can be used for the diagnosis of trypanosomiasis using patient serum polyclonal antibodies.

We will create Antigen-43 and Ice Nucleation Protein constructs, to be expressed on surface of E. coli, that carry an additional sequence of amino acids referred to as a ‘mimotope’. A mimotope (mimic epitope), functions as a target for disease-specific antibodies in the blood, which may be present due to an active immune response to a pathogen. A green fluorescence response indicates a positive disease diagnosis.

Antigen-43 (Ag43) and Ice Nucleation Protein (INP), are autotransporter proteins that exist endogenously on the surface of E. coli; they can transport large peptides for cell-surface display. We have concentrated on creating ‘BioBrick’ versions of these for submission to the iGEM 2014 repository.

The Ag43 has had its PstI restriction sites removed to make it BioBrick compatible, the two Beta-hairpins removed to prevent cellular auto-aggregation. A BglII/HindIII restriction site has been added for easy future insertions of desired sequences. We have also introduced a FLAG-tag to create a demonstrable ‘proof of concept’ for antibody aggregation-based detection. We intend to also create a His-tag version.

We began with an INP-YFP (Yellow Fluorescent Protein) BioBrick created by a previous team. To this we added a BglII/HindIII restriction site. We also introduced a FLAG-tag and His-tag to this site in 2 separate versions.