Team:GreatBay United/Description

GreatBay_United

GreatBay_United

Description:

Project Inspiration

GreatBay_United is a team derived from a newly-developed coastal city where advanced technology collides with enthusiasm of protecting marine environment and biodiversity. Members of our team are a group of students who have strong bonds with coastal animals as well as ocean. That was how we came up with the idea of relating synthetic biology with the protection of horseshoe crabs, which is an incredibly meaningful animal but endangered...This year, GreatBay_United is aiming to use the power of synthetic biology as well as our smart brains to save this critical creature and maybe the future of human beings.

At present, bacterial endotoxin infection has become a major problem in medical industry. The main chemical substance of bacterial endotoxin is lipopolysaccharide. The harmfulness of gram-negative bacterial infection is mainly due to the release of endotoxin. After entering the body, bacterial endotoxin can successively induce the production and release of inflammatory mediators such as cytokines and adhesion molecules, resulting in endothelial cell injury and barrier function change, leading to systemic inflammatory response syndrome and sepsis. In severe cases, it can lead to hypotension, toxic shock, meningitis, disseminated intravascular coagulation (DIC), acute respiratory distress syndrome (ARDS), multiple organ failure (MOF) and even death. The approachable method of detecting endotoxin has become significantly important.

Now there is an efficient and convenient method to detect the presence of bacterial endotoxin - LAL test. From the moment it was discovered, limulus agent has been widely used in a variety of possible detection of bacteria in the field. The demand for limulus reagent has been increasing over time. LAL testing is required for every drug licensed by the US Food and Drug Administration (FDA). Limulus reagent is extracted from limulus blood. The increase of limulus reagent demand undoubtedly means the increase of limulus blood demand. Therefore, the blood of horseshoe crab plays a pivotal role in the modern biological industry, and its value is irreplaceable in a period of time. Once the horseshoe crab disappears, it will have a huge impact on the modern biological industry.

Unfortunately, existing horseshoe crabs are not living well. On the one hand, environmental pollution leads to less and less living environment for horseshoe crab. On the other hand, even though horseshoe crab has become a protected animal, some people still hunt it illegally, and meanwhile the demand for horseshoe crab reagent only increases as goes by.


Attention of problems and the huge testing requirements are in stark contrast with the lack of similar detection technology, the scientific community‘ knowledge about how to artificial limulus blood detection of bacterial endotoxin, although there are relatively complete substitute limulus reagent, but the bacterial endotoxin test method such as restructuring c factor method should be to hours of incubation time. Moreover, it is not mature enough and it poses a threat to drug safety and requires a long detection time. The recombinant C factor reagent‘s preservation also depends on the specific temperature, and needs to be operated in the laboratory, which means it is limited by the site of use. If we can develop the most efficient and fast artificial limulus reagent, fill the vacancies of bacterial endotoxin detection technology in industry, not only can reduce the probability of death due to infection of bacterial endotoxin, but also can provide a simple treatment of bacterial endotoxin for later quantitative validation of the method, also save an endangered species in the future, is of far-reaching significance.

The goal of GreatBay_United is to create a practical artificial LAL which can hopefully replace LAL by using protein characterization of engineered strains of synthetic biology. Once LAL is replaced, there will no longer be such great demand of horseshoe crabs' blood. We may actually benefit human being and as well save an endangered species. We plan to design multiple systems and put them together to construct a fully functional artificial LAL.

Design

Based on how LAL functions, the overall process can be separated into three major parts - detecting, cascading and agglutination. Therefore our team designed three systems which match these parts and make sure they function well enough to match the actual LAL.

1. 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. PSA protease is the trigger switch of the cascade reaction. 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 action domain was exposed, thus activating the downstream cascade reaction.

2. Cascading system

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.

3. Agglutination system

The agglutination system is based on yeast surface display. A portion of the yeast cells will express GFP and their expressed GFP will be suppressed by linker linked to anti-GFP, while the other portion of the yeast cells will express anti-GFP. The HCV from activated cascade system will cut substrates placed in linker to activate GFP, which can be combined with anti-GFP expressing yeast cells to achieve cell level agglutination through protein level binding, and achieve the visualization of LPS testing results.