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

We created Antigen-43 and Ice Nucleation Protein constructs, to be expressed on the 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. Our system carries two different antigens that will allow the detection of an immune response to trypanosomes with minimal false positive results. The antibodies from the serum will are attached to a poly-lysine coated cuvete, or other appropriate surface before the introduction of the E.coli. This allows communication between a Quorum Sensing (QS) sender and receiver gene that causes the production of GFP when there are enough cells of each within close proximity to each other. 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. Auto transporters are proteins that are capable of localisation to the cell surface and forming their own protein complex to allow surface expression without the use of other proteins. One interesting feature of some autotransporters is that they have the capability to contain other large peptide sequences for cell-surface display without altering the final structure of either the autotranspotrer or the passenger. 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, with two Beta-hairpins removed to prevent cellular auto-aggregation. A BglII and HindIII restriction site has also 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.

For our INP Biobrick we began with an INP-YFP (Yellow Fluorescent Protein) BioBrick created by the Edinburgh 2010 iGEM team. To this we added a sequence consisting of BglII, a FLAG tag and His tag (in different constructs), followed by a HindIII restriction site.