To recap... In 2021, we made the submission of these two parts to the Registry:

In 2022, we made use of these parts to perform experiments.

In synthetic biology one of the main principles is the combination of parts to generate a new one, a biobrick, which will produce a new compound/product. Thus, in our project we used coding sequences of the PLA2 protein present in jararaca, crotalus and jararacussu venom, as well as the coding sequence of the jararaca 𝛾PLI, which can be used by future teams. For this, we performed the tests, in laboratories using Escherichia coli bacteria for the expression of the protein of interest and expression of parts of the venom causing the necrotic effects to perform the inhibitory assay, proving the effectiveness of the project.

BBa_K4091024 is a myotoxic PLA2 from Bothrops jararaca, and we intend to use it to better understand the action of this important component of snake venom. BBa_K4091069 is a phospholipase A2 inhibitor, and we apply it to reduce the myotoxic actions of the snake's PLA2. In order to aid purification, we added a His-TAG and, for cleavage, a TEV-site.

Initially, we performed a search for the cDNA sequences for γPLI and PLA2 from Bothrops jararaca using the NCBI database. Our proteins come from snakes, so they are eukaryotic organisms and to express them we used E. coli strain BL21 DE3 as a chassis, the choice of this microorganism for expression was based on the fact that 𝛾PLI hasn’t post-translational modifications such as glycosylation, and also because E. coli is a well-known and fast-growing microorganism. For the verification and removal of possible signal peptides we used the SignalP 5.0 tool from DTU Bioinformatics. The Expasy translation tool was used to ensure that our sequence would express a functional protein after checking and removing any restriction sites for EcoRI, NotI, PstI and SpeI from the gene.

In this project we developed a circuit with a T7 promoter with RBS (BBa K525998), this is because T7 works very well with E.coli BL21 DE3, the chassis used in the project to express the 𝛾pli protein. In addition, our team already had a protocol for using this technique. Furthermore, we used a double terminator (BBa B0015) because of its safety, since it is already widely used for the expression of therapeutic proteins, and because it has a good compatibility with our chassis. As a vector we used the PSB1C3 present in the iGEM kit, which is resistant to the antibiotic chloramphenicol; we chose this vector because it is widely used by several teams and also because it is a standard iGEM vector.

In 2022, we return with the following new parts:

Jararhagin highly hemorrhagic P-III SVMP (metalloprotease) class protein from Bothrops Jararaca venom. It has a molecular mass of 52 kDa, which is considered high. In the catalytic domain is the zinc-binding sequence HEXXHXXGXXH and the Met-turn structure, which helps stabilize the three histidine residues involved in catalysis (Bode et al., 1993; Stöcker et al., 1995). In the disintegration domain, there is conservation of the cysteine residues in the same position as those found in RGD, but with replacement by an ECD sequence, common to other P-III SVMPS. That is, it has the disintegration domain (BJARNASON; FOX, 1995). The cysteine-rich disintegrin domains have high similarities to domains found in ADAMs (Paine et. al., 1992).

BJ46a, which can be taken from the plasma of Bothrops jararaca, is an acidic glycoprotein, with molecular mass of 46 kDa, capable of inhibiting the activity of the metalloproteases jararhagin and atrolysin-C by forming non-covalent complexes with such SVMPs. Studies indicate that despite the presence of the glycidic portion in ISVMP, this portion does not present a direct relevance for its proteolytic activity (unlike other inhibitors, such as DM43), which indicates that the interaction regions between the inhibitor and toxin occur in the protein portion of the glycoprotein.

As described in Contribution, we performed the part design, optimizing codons for P. pastoris and E. coli, and designed primers for expression with each of these microorganisms.

1. M.J.I. Paine, H.P. Desmond, R.D.G. Theakston, J.M. Crampton Purification, cloning and molecular characterization of a high molecular weight hemorrhagic metalloprotease, jararhagin, from Bothrops jararaca venom. Insights into the disintegrin-like gene family J. Biol. Chem., 267 (1992), pp. 22869-22876.


2. BJARNASON, J. B.; FOX, J. W. Snake venom metalloendopeptidases: Reprolysins. Proteolytic Enzymes: Aspartic and Metallo Peptidases, p. 345–368, 1995.


3. W. Bode, F.X. Gomis-Ruth, W. Stockler Astacins, serralysins, snake venom and matrix metalloproteinases exhibit identical zinc-binding environments (HEXXHXXGXXH and Met-turn) and topologies and should be grouped into a common family, the ‘metzincins’ FEBS Lett., 331 (1993), pp. 134-140.


4. BASTOS, Viviane A. et al. Natural Inhibitors of Snake Venom Metalloendopeptidases: history and current challenges. Toxins, v. 8, n. 9, p. 250, 2016.