Team:Baltimore BioCrew/Improve

Improved Parts

The Baltimore BioCrew’s research into projects relating to blood coagulation led them to research about Lethbridge HS’s 2016 iGEM project. We observed that their team had previously created a construct using a serine protease from the Cerastes cerastes (Desert Horned Viper) called “Cerastotin” and that it had the ability to coagulate mammalian blood at a faster rate than usual.

However, a few modifications could have been added to their construct to overall improve its functionality. The first improvement made to the Cerastotin construct was an overall codon optimization specifically optimizing it to work better in E. coli.

Next, we decided to implement in our optimized construct was a 6xHis tag. This tag gave us the ability to purify our protein through a Nickel-affinity column, and thus more effectively isolate our venom for various experimentation purposes including testing the potency of our venom, as well as to compare its overall level of regulation, as well as to test whether or not it can be purified.

Lastly, we replaced their constituent promoter with the Lacl promoter because the Lacl promoter is more regulated than that used by the Lethbridge construct. Another additional improvement that the Lacl promoter gives us is the possibility of inducing it using either lactose or IPTG.

Overall, we made three separate constructs for every step of the improvements, and we renamed our constructs from "Cerastotin" to "Cerastocytin":

Cerastocytin Constructs:
Optimized: BBa_K2893001
His-Tag: BBa_K2893002
His-Tag and LacI Promoter: BBa_K2893000