Unfortunately, our team couldn't perform the wet-lab for the Honorato 2.0 project because both our iGEM 2022 kit and our parts for synthesis did not arrive in our lab due to transportation problems. In addition, all the iGEM 2021 kit were degraded, which delayed our project and prevented us from completing it. Despite this, we used the tools that were available in our lab and could make a big part of the wet-lab with the synthesized parts that we already had from the 2021 project. Even with the delay, the "Engineering Success" requirement for Honorato 2022 project was mostly reached from the mathematical modeling as shown below, complementing the wet lab work and concluding the requirement.
Ophidic accidents represent a serious public health problem in tropical countries, due to the frequency with which they occur and the mortality they cause. In Brazil, jararacas are responsible for 70% of the accidents, which can range from injuries to hemorrhages or necrosis of the affected sites.
Being a complex mixture composed largely of enzymatic proteins, such as phospholipases, metalloproteases, oxidases, acetylcholinesterases, lectins, and peptides, an important protein is directly related to necrosis: the main short-term effect of the snake bite. Currently, the treatment for this type of accident is the antiophidic serum, produced from large animals, specifically the horse. The production of this serum goes through several steps, beginning with the extraction of the snake venom and continuing with the transformation into antigen. Small doses of this antigen are applied to the horse's body in order to sufficiently stimulate the production of antibodies in the animal's blood. Finally, its plasma is collected and undergoes a complex industrial processing to obtain the serum.
Currently, the treatment for this type of accident is the antiophidic serum, produced from large animals, specifically the horse. The production of this serum goes through several steps, beginning with the extraction of the snake venom and continuing with the transformation into antigen. Small doses of this antigen are applied to the horse's body in order to sufficiently stimulate the production of antibodies in the animal's blood. Finally, its plasma is collected and undergoes a complex industrial processing to obtain the serum.
The main objective of the project is to study the production of a protein capable of inhibiting one of the many toxins present in B. jararaca venom, specifically an enzyme that is primarily responsible for the myonecrosis effect: phospholipase A2. The inhibition will be done using the model bacterium Escherichia coli and using recombinant gene technology to express the 𝛾PLI protein (𝛾 phospholipase inhibitor), the natural inhibitor found in the blood serum of venomous snakes such as the genus Bothrops and Crotalus. Thus, with the inhibition of phospholipase A2, we intend to control more effectively one of the most harmful effects that require urgent medical treatment. Additionally, producing this protein through synthetic biology can reduce the use of animals to obtain the anti venom serum, also avoiding the occasional death of animals during the traditional process of obtaining the serum.
From the development of the 2021 Honorato Project, as a continuation of the research, our team studied the hemorrhagic effects of jararaca venom, specifically, the action of Jararhagin. Being a metalloprotease present in Bothrops jararaca venom, when it enters the bloodstream, it inhibits platelet aggregation introduced by collagen and ristocetin, interfering directly in the blood coagulation process and, therefore, causing consequent hemorrhagic lesions (Wang & Huang, 2002). Moreover, it also has an effect of inhibiting the adhesion of endothelial cells to collagen-coated plaques, thus leading to the weakening of the blood vessel wall and the collaboration of these bleeding lesions.
As a way to remedy this immediate problem of ophidisms caused by Bothrops snakes, the alternative to the antidropic serum that we propose, under the same interpretation of the problem described in the 2021 project, is the recurrence, through synthetic biology, of a natural inhibitor for Jararhagin.
Coming from the blood plasma of the Bothrops jararaca snake, BJ46a is a natural inhibitor, classified within the snake venom metalloproteinases inhibitors (SVMPI) (BASTOS, 2014). But how do we study the implementation of this inhibitor as a treatment for the problem we study? First, we evaluate the structure and physicochemical characterization of BJ46a and Jararhagin molecules; then, we research the mechanisms of action to finally confront them in inhibitory assays against Jararhagin.
In developing the modeling, we evaluated the proteins structurally and dynamically, i.e., looking for the closest crystal structure and predicting the best protein stability in vials, with the goal of a more feasible application to social reality. The modeling performed is homology, but why did we choose it? It is currently the most accurate computational method for generating reliable structural models, since it builds three-dimensional models of protein structure using experimentally determined structures of related family members.
The idea is that a structural description of how the enzyme-substrate interaction happens for the analyzed protein pair. Both the gene sequencing and the activity interface were reviewed in order to follow the simulations.
How would the idea of gene expression be configured? The assembly of synthetic gene circuits is widely used in modulating the expression and control of genes in the metabolic pathway of microorganisms to synthetically develop a biological organism with predictable and regulable metabolism.
Figure 1 - Abstraction hierarchies for synthetic biology from parts, circuits to genomes.
Source: (CHEN et al., 2018)
Then, the biosynthetic circuit we developed used the PPIC9K vector, under the Gibson Assembly methodology. Then, under the 3A Assembly methodology, we built a circuit for production and secretion of the protein in E. coli bacteria, as well as a KillSwitch circuit for E. coli, also following the 3A Assembly methodology.
In parallel, we needed to fetch our initial data of our Jararhagin protein and our BJ46a inhibitor, searched in the NCBI database for submission to SWISS-MODEL, a fully automated and accessible protein structure modeling server, via the Expasy web server, or the DeepView program (Swiss Pdb-Viewer). Following this, modeling of the BJ46a inhibitor was performed by the Modeller program, a homologous modeling development software that uses the python language in its execution, whose obtained parameters were recorded.
Figure 2 - Modeling of protein BJ46a
Source: AUTHOR - Modeller and Chimera
Next, a mathematical analysis of the parameters obtained was performed in order to obtain the best possible structure, according to the scoring parameters analyzed.
Subsequently, the modeled structures and molecular docking tools were used to analyze the inhibitory interaction between the Jararhagin protein and the BJ46a inhibitor. To perform the docking, first the servers to be used to trace the interaction between the two proteins were selected and, consequently, the parameters obtained were recorded for further analysis. We used the servers ClusPro, PatchDock, HDock and PyDock. It is noteworthy that the results obtained were corresponding between the servers and converged with the data obtained in the articles.
Finally, molecular dynamics was studied to predict the stability of the inhibitor, a protein, in aqueous media in order to obtain a better fit of the model to reality. In this part, the GROMACS software and the CHARMM-GUI server were used to perform the simulations, according to the characteristics of the BJ46a protein.
For both software, there was agreement on stability conditions, with additional optimizations of the drug storage for filling into cubic containers, as presented in the CHARMM-GUI results.
Thus, as we did all the research regarding the structural modeling, the study of the kinetic modeling of our project was also done, because a good modeling of the kinetics enables us to understand the mechanisms of inhibition of our protein with our inhibitor, saving a lot of time and also raw material throughout the phase of laboratory experiments.
Initially we searched several literatures and understood very important concepts for us, such as the fact that proteins are not allosteric, so they can be modeled following the Michaelis-Menten mechanism (NEVES-FERREIRA et al., 2015). Furthermore, proteins interact by forming a non-covalent, i.e. reversible, complex in which the inhibitor competes with the substrate for the (competitive) binding site (VALENTE et al., 2001).
With this in mind, we started with the stoichiometric calculation in order to have a molar and mass proportion for each inhibition range that we will evaluate. Concomitantly, we tested BJ46a against atrolysin-C and against Jararhagin by inhibitory assay, using the fluorogenic substrate of Abz-Ala-Gly-Leu-Ala-Nbz.
As previously stated, we used the Michaelis-Menten mechanism to obtain an equation that described the inhibition between the two proteins in our study and, by doing so, we expected to obtain a non-covalent enzyme-inhibitor complex with a substrate in the medium. For this, we used the experimental data obtained from the inhibitory assays and from literature regarding the inhibitor BJ46a, two metalloproteinases that have great genetic similarity with the metalloproteinase Jararhagin and the gelatinous substrate Enzchek.
After that, we did a linearization of the data we obtained by Lineweaver-Burk, and built a graph with the already linearized data, and extracted it using the GetData software. Later, we used this data to construct a mathematical resolution, which will be demonstrated later. Then, using the GeoGebra and Scilab software, we plotted a logarithmic curve that represented the time traveled in relation to the remaining substrate.
However, our modeling was not only based on theoretical calculations. We also performed inhibitory assays to analyze the interaction of BJ46a with different metalloproteinases from snake venoms, which had already been shown in previous studies (there is a proven interaction with the metalloproteinases Jararhagin and astrolysin-C), with no complex formation with Jararhagin-C and indicating that the domain is fundamental for the inhibitor-toxin interaction. Thus, following the experiment proposed by Bastos (2014), we selected SVMPs from three snakes, atroxlysine (from Bothrops jararaca), leucurolysin-a (from Bothrops leucuros) and BaP1 (from Bothrops asper).
Evaluating the panorama of the modeling performed, diverse knowledge was developed by the research. From the knowledge regarding synthetic biology, which are the genetic sequencing, the enzyme-substrate and inhibitor interaction, the kinetics of the enzyme catalytic activity, to the knowledge of computing and engineering, with the use of software, programming languages, evaluation of mathematical parameters, and circuit construction.
From the project, the interaction of the chosen natural inhibitor brought values that reach, by Valente (2001), 91% inhibition of the enzyme activity, under the 1:1 ratio between enzyme and substrate (BJ46a). Moreover, the storage conditions, with the optimal temperature and pH of the protein, obtained via computer simulations, allowed the knowledge of the necessary interactions of the drug, as a product destined for distribution and application in society.
With the construction of the modeling described, we developed improvements and optimizations of the genetic sequencing congruent to our worked proteins, as well as their respective primers. There was the optimization, through software, with the addition of start and stop codons manually, besides the insertion of the promoter and terminator, of Jararhagin and BJ46a for Pichia pastoris and for Escherichia coli. In addition, the amplification primers for these proteins were made for the expression organisms, with the primer range optimized for the vector used in the methodology described.
To better understand the issue to which we are facing, we must firstly understand why ophidian accidents are among the neglected tropical diseases (WORLD HEALTH ORGANIZATION). The venom secreted from snakes is considered Nature's most complex mixture, since those animals have evolved to maximize the efficiency of its venom. Traditionally, the production of the antivenom begins with the inoculation of the poison in large animals. The concentration of the substance applied is gradually increased through weeks or months until the animal is able to synthesize enough antigens to be purified in the next steps. Some blood samples (a few liters) are taken from the animal and then centrifuged to separate the plasma from the other blood elements. The plasma contains the antibodies that are going to inhibit the enzymes found in the snake venom. This fraction of the blood is then purified and some chemical compounds are added to guarantee a satisfactory stability as well as “shelf time” (DINIZ, 2016). This process yield is high and it is also relatively cheap, so there were not many reasons to search for more humane and more technologically advanced methods, sparing the animals from suffering during this process. It is also a disease predominantly found in third world countries, which has naturally less resources to do this kind of research.
Among the proteins found in snake venom, there are two that we decided to focus our study on: The Phospholipase A2 and the Jararhagin, enzymes responsible for the myonecrotic effect and the hemorrhage, respectively. It is important to remember that our project does not inhibit the full synergetic effect of the snake venom, since there are multiple enzymes that are not even fully characterized by the literature yet (VIEIRA, 2010). Since the traditional way of producing the antivenom is through large animals, we decided to take the first step towards a fully synthetic snakebite serum, so that we can stop using animals such as horses to inhibit the venom. To do so, we investigated why snakes are immune to its venom, which is the presence of a protein inhibitor in the snake blood plasma: the 𝛾PLI (Phospholipase inhibitor) and BJ46a. Funnily enough, it all started with a silly question: What would happen if a snake bites its own tail? In Brazil, the serpent genus which causes most accidents is the Bothrops, more specifically, in the southwest of Brazil, the most dangerous serpent is the Bothrops jararaca, which is the main motivation for the Honorato Project.
WHAT COULD BE OUR CHASSIS?
To test the possibility of expressing those proteins and modeling its kinetics, we used synthetic biology to design a biological circuit and modify Escherichia coli cells (BL21 DE3) into expressing our inhibitors as well as its enzymes.
Firstly, to achieve our goal to express our target proteins (𝛾PLI and PLA2), we requested oligonucleotides to amplify our genes via the PCR (Polymerase chain reaction), which would work for both gene because we assembled it with the same terminators and promoters.
We assembled the parts together using a modified 3A assemble method to better fit into our target protein, lab costumes and capabilities. As for the chosen parts, we used a composite promoter T7 with a RBS (BBa_K525998) and a double terminator (BBa_B0015). We chose those parts because it is known by the literature and previous igem teams that both the promoter and the terminator work exceptionally well in our chassi, the E. coli BL21 DE3. We added restriction sites between the parts in case that if we could not express our protein properly, we could try other parts in the distribution kit. The biological circuit was connected with PSB1C3 as its plasmid backbone. Our plasmid was cloned in E. coli Turbo and the plasmid was minipreped using a Wizard Plus SV Minipreps DNA Purification System. Then, after the proper enzymatic digestion and ligation, we transformed into E coli BL21 DE3, our expression chassi. Our transformations were confirmed through colony PCR followed by gel electrophoresis. Once our proteins would be properly synthesized and purified, we would have done the inhibitory assay to which in combination with our kinetic model, we would obtain the value of several parameters that describe the behavior of the reaction. Finally, we would have done the design of another snake venom component to do an analog procedure: the jararhagin and its inhibitor BJ46a. We choose E.coli instead of a eukaryotic chassi because our protein has few glycosylations and it is simpler to do the experiments, we also have bibliographic evidences that support the fact that it can be achieved with this prokaryotic organism, even though it cannot do any post transcriptional modification. (SILVA, 2017). As for the second part of this project, the expression of the jarahragin and its inhibitor should be done with Pichia pastoris, since those proteins are a bit more complex and thus need glycosylation and other post transcriptional modifications.
Unfortunately, the IGEM 2022 distribution kit did not arrive, so we had to use the IGEM 2021 kit to get our plasmid, which was completely degraded as far as we tested. Given our time and possibilities, we have completed the engineering cycle effectively, designing our circuit, expressing our target protein, enhancing our method and learning new information to be applied in future experiments like the implementation of our concept of the jarahragin and BJ46a expression in P. pastoris.
So far, we are in the process of expressing and purifying our proteins, we achieved that much without a proper distribution kit. We estimated the size of our target proteins (PLA2 and 𝛾PLI) through the expasy software, it is possible to observe bands of proteins in the regions around 42 and at 50 kDa, which suggests that the expression of such proteins were successful.
Figure 1 - SDS PAGE result for Phospholipase A2
Source: Author (2022)
Figure 2 - SDS PAGE result for 𝛾PLI
Source: Author (2022)
Figure 3: Electrophoresis E. coli positive colonies with parts: 𝛾PLI and PLA2
Source: Author (2022)
Figure 4: Electrophoresis with positive colonies of our chassi with the parts: 𝛾PLI and PLA2 jararaca.
Source: Author (2022)