(28s) Multivalent Anchoring of Cell Wall Binding Domains By Using Split Fluorescent Proteins

The transmission of bacteria often leads to infections and diseases resulting in the need for rapid and highly sensitive bacterial detection methods. Lytic enzymes are typically exceptionally specific to a target bacterium. For lytic enzymes that target Gram positive bacteria, they generally consist of a cell binding domain (CBD), which binds to the peptidoglycan layer of target bacterium, and a catalytic domain that cleaves the peptidoglycan, degrading the cell wall and killing the cell. Importantly, the CBDs can provide for a nondestructive detection method. However, binding sites on the cell surface are often limited, with results in low detection sensitivity. To resolve this issue, we developed a split green fluorescent protein (GFP) construction in which multiple GFPs are linked together and fused with a specific binding domain. We designed two components, CBD-GFP beta strands 1-10 (GFP_1-10) and a chain of GFP₁₁ strands, which were recombinantly expressed and complemented in E. coli using in vivo assembly. Flow cytometry showed higher fluorescence intensity as the number of linked GFPs increased. Surface plasmon resonance (SPR) was used to quantify the binding kinetics between these proteins and their target bacterium cell wall. By utilizing streptavidin/biotin interactions, we coupled the split GFP-CBD system with horseradish peroxidase (HRP). Upon addition of substrate, a visible signal is generated, which to increases detection sensitivity. HRP activity followed the same trend as flow cytometry, such that longer GFP chains with higher bound HRP resulted in greater catalytic activity. When applied to a surface, we saw a reduction in the limit of detection (LOD) within 30 min to 2.5 x 10³ CFU/cm². We proceeded to expand this approach using a red fluorescent protein (RFP) coupled with another lytic enzyme binding domain and we obtained a similar outcome as with GFP. Employing both GFP and RFP CBD fusion proteins, multiplex detection was obtained on two specific bacteria simultaneously. The coupling of CBDs to multiple split fluorescent proteins enables multivalent anchoring of an enzyme that generates an amplified signal of the presence of specific bacteria on surfaces and solutions.