Team:GreatBay United/design

GreatBay_United

Design:

Based on how LAL works to achieve the visualisation of the detection results of endotoxin, we sperated our aritificial LAL into threee parts: The detection system to activate the following system when endotoxin is present, the casecade system to magnify the signal and the agglutination system to achive cell-level agglutination.

Detection system

The detection system was composed of PSA protease and fusion proteins consisting of LPS binding domain and LPS mimic domain of specific bacteria. LPS Binding domain and LPS mimic domain are located at the N-terminal and C-terminal of PSA protease respectively. Due to high affinity between LPS binding domain and LPS mimic domain, The fusion protein in the detection system was folded into a ring, and the PSA protease active domain was obscured, which could not trigger the cascade reaction. When LPS was present in the reaction environment, LPS binding domain was competitively combined with LPS mimic domain, the fusion protein was allosterized and PSA protease actice domain was exposed, thus activating the downstream cascade reaction.

Cascade system

PSA protease is the trigger switch of the cascade reaction. We use allosteric protein as the switch of the cascade system, and HCV (the final product of the cascade system) as the switch of the agglutination system, combining the detection of allosteric protein for LPS with the agglutination system through the cascade system. The cascade system will be composed of TVMV protease and HCV protease, amplifying the signal through the mechanism of cascade protease cutting and protease activation, realizing the function of greatly improving the detection sensitivity.

Agglutination system

The agglutination system is based on the yeast surface display technology. Some yeast cells will express green fluorescent protein, while other yeast cells express anti-GFP. HCV protease of the cascade system will cleave the substrate placed on the linker to activate GFP, which can combine with yeast cells expressing anti-GFP to achieve cell-level agglutination, visualizing LPS detection results.