The purpose of our project originated after a brainstorm discussion on what problems most affected Baltimore City citizens. We went through ideas such as air pollution and curing cancer, and presented our project ideas to each of our team members. However, the projects that really stood out to our team were the clot making and clot breaking.
The clot making team focused on treating trauma injuries, such as those caused by gun violence. We knew that with Baltimore City having one of the highest murder rates per capita in the country, it was important to figure out a way to lessen the rate of murders through treatment. We noticed that current bandages and sponges with coagulation enzymes were expensive, and also wanted to make sure that our final product would be cost effective for Baltimore citizens.
The clot breaking team looked at diseases that were caused by clot formation, such as strokes and heart disease. Other than being known for violence, Baltimore has a high rate of heart disease which often goes untreated. The clot breaking team wanted to use a product that was more cost effective than tPA but had the same level or better level of efficiency.
After the clot making and clot breaking team presented their ideas, the entire Baltimore BioCrew team was split between picking the clot making or clot breaking team for our 2018 iGEM project, so we picked both.
Once we had our projects decided, we began to conduct research on how to design our constructs.
The clot making team looked at ways that previous teams have coagulated blood. The first team that we came across was the 2016 Lethbridge High School team, who used Cerastotin (Horned desert viper) as their enzyme for coagulation. We saw that they were successful and decided to contact Dr. Brian Dempsey, who gave us more information about their team’s constructs. Upon hearing about these constructs, we decided to make three constructs that included their original construct. For all of the constructs, we codon optimized for E.coli. One construct included a His-tag for nickel column purification and a LacI promoter for tighter regulation and inducibility. The second construct included a His-tag while as the third was just the codon optimized construct. We named these constructs “Cerastocytin” after not being able to pronounce Lethbridge’s construct.
After being interested in the possibility of using snake venom to coagulate blood, we looked into other possible enzymes that could be incorporated into other constructs. We researched multiple different snake serum proteases, such as Batroxobin and Russell’s Viper Venom. However, we ultimately decided on Russell’s Viper Venom since Batroxobin has carbohydrates that would not work well with E.coli. Also, Russell’s Viper Venom has been tested in numerous laboratories and has been able to speedily and effectively coagulate mammalian blood.
We decided to design our constructs similar to how we created our Cerastocytin constructs. We created three constructs and codon optimized all three of them. The three differing constructs were the exact same as the Cerastocytin constructs, except that instead of having the Cerastotin enzyme, we replaced it with the Russell’s Viper Venom serine protease.
Although the most commonly talked about cause of death in Baltimore are shootings, the clot breaking team decided to focus on the actual most common cause of death in Baltimore: heart disease. More specifically, diseases that were caused by clot formation, such as strokes or heart attacks.
Finding a method of decoagulation was not difficult, as the clot breakers researched what the human body naturally uses, as well as what is clinically used, to decoagulate blood: tPA. Potential issues were brought up with the folding of the tPA, and as such, the clot breakers sought out to find another more consistent option. Ironically following the trend of the project being related to snakes, the clot breakers found a relative of tPA (reptilase) that is shorter with fewer disulfides, and thus was more likely to fold correctly. These two genes were used to design two constructs. On top of that, the clot breakers added a T7 promoter to both genes to better regulate their expression (though a construct was designed using a standard constituent promoter for a variation of the standard tPA construct).
To test our constructs efficiency, we planned to transform our constructs into DH5∝ E.coli cells, run a colony PCR screening, send them off for sequencing, and run protein gels.