Team:USP-EEL-Brazil/Engineering

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BEYOND SYNTHETIC BIOLOGY

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In the first research, our team decided to focus on a big problem related to our community. At the first moment we gathered different ideas, that vary from, Bioenergy, to therapeutics and then analyzed them according to what we could do in our University, and the impact in our society.

Came to our mind, the problematic related to ophidic accidents, and all the difficult for the serum production and the access to the treatment. The findings about the high incidence of ophidic accidents in Brazil, mainly caused by the Bothrops genus, and also by analyze the venom composition, we realize that focusing on the Phospholipase A2, was the best path to follow on this project, due to its mionecrotic effect and synergy among other enzymes.

Our design was made with a T7 Promoter, for a constitutive expression and a compatibility with our E. coli, just like our (Ribosome Binding Site BBa K525998). For our coding sequence, we have chosen to use a sequence of a Gamma-Phospholipase A2 Inhibitor from Bothrops jararaca, which we cutted off the signal peptide sequence and optimized for our chassis. An important fact is that with this inhibitor, is that due to the similarity of the phospholipase A2 from B. jararaca and others snake from Bothrops genus and also Crotalus, have a significant identity, therefore, we can expect that the Inhibitor should work against Phospholipase A2 from other Bothrops and some Crotalus species due to its high similarity.

At final we’ve chosen a double terminator (BBa_B0015), built with a T7 terminator and an E. coli one, to ensure the end of transcription.

At the Modeling, we first predict some models using the Modeller software where a python code was written for model the structure, followed by using the I-Tasser WebServer, which was able to gather the best 10 templates and predict the best model.

We have validated our model, by evaluating the C-Score and the TM-Score, which showed us good confidence. Also the inhibition modeling we did, shows us that the inhibition increases with the raise of PLI concentration.

By working on structure and inhibition modelling, we learn about sequence alignment, python, model evaluation and scores, and also about enzymatic kinetic modeling.

Building the model by homology modeling using Modeller we reached a good model by using 3 templates found with the support of the Swiss-Model web server. However, we decided to use the i-Tasser web server along with LOMETS, to build a better model with the most accurate templates. With the inhibition modelling, we have a perspective to achieve a precise concentration, since now we have the tendency of inhibition that increases with the concentration of the PLI.

For future projects we expect to have a docking simulation, to see in molecular and structure levels how inhibition with γPLI from B. jararaca works. But mainly to improve and support ophidic accident therapeutics.

Serino-Silva C, Morais-Zani K, Hikari Toyama M, Toyama DdO, Gaeta HH, Rodrigues CFB, et al. (2018) Purification and characterization of the first γ-phospholipase inhibitor (γPLI) from Bothrops jararaca snake serum. PLoS ONE 13(3): e0193105. https://doi.org/10.1371/journal. pone.0193105

Email: symb-lab@usp.br