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Parts

Parts_ CHELO

Overview

 Our project focuses on detecting proteins with abnormal expression levels or activity due to disease, with leukemia chosen as the primary target. Through model screening, CD97 was identified as our detection target. Using a predictive system, two short peptides, 2BOU-2 and 2BOU-3, were selected for their ability to bind to CD97. Structure alignment showed that the structures of these peptides are similar to the binding site of CD55 which we found from literature on CD97. As a result, we linked CD55, 2BOU-2, and 2BOU-3 to a transmembrane protein system, BELO, (Lpp-OmpA-GS Linker) to display them on the cell surface. We then monitored the changes in electrical current upon binding to CD97, comparing the results across varying concentrations of CD97 using a chip-based detection system.

Natural binding protein of CD97

 After identifying CD97 and the predicted short peptides, we conducted sequence alignment and reviewed literature, discovering that CD55 is a molecule capable of binding to CD97. As a result, we expressed CD55 on the cell membrane with the expectation that it would successfully bind to CD97.

Predicted Peptides

 Using structural prediction modeling, we generated short peptides capable of binding to CD97. These peptides were transported to the cell surface via the Lpp-OmpA-GS Linker, and the changes in electrical current upon binding to CD97 were then measured.



Composite Parts

Lpp-OmpA-GS Linker-GFP-His (BBa_K5151009)

composite part BBa_K5151009
Figure 1. The composite part BBa_K5151009 contains PLac promoter, RBS, Lpp-OmpA-GS Linker, GFP and 6X His tag.

We attached GFP and 6X His tags to the Lpp-OmpA-GS Linker, and confirmed their surface expression through ELISA analysis.

Lpp-OmpA-GS Linker-CD55 (BBa_K5151010)

composite part BBa_K51510
Figure 2. The composite part BBa_K5151010 contains PLac promoter, RBS, Lpp-OmpA-GS Linker and CD55.

CD55 was expressed on the cell surface using the Lpp-OmpA-GS Linker. CD97's receptor is CD55, which was identified from literature as a protein capable of binding to CD97.[1]. After binding to CD97-His, Anti-His-HRP and TMB were added, and the resulting changes in electrical current due to electron transfer were measured.

Lpp-OmpA-GS Linker-2BOU-2 (BBa_K5151006)

composite part BBa_K5151006
Figure 3. The composite part BBa_K5151006 contains PLac promoter, RBS, Lpp-OmpA-GS Linker and short peptide, 2BOU-2.

We introduced Generative AI to design short peptides capable of binding to the potential biomarker, specifically 2BOU-2, with the expectation that it would bind to CD97-His.

Lpp-OmpA-GS Linker-2BOU-3 (BBa_K5151015)

composite part BBa_K5151015
Figure 4. The composite part BBa_K5151015 contains PLac promoter, RBS, Lpp-OmpA-GS Linker and short peptide, 2BOU-3.

We introduced Generative AI to design short peptides capable of binding to the potential biomarker, specifically 2BOU-3, with the expectation that it would bind to CD97-His.



Reference