One of the contributions of our team for 2022 iGEM consisted in the optimization of gene sequences for both Pichia pastoris yeast and Escherichia coli bacteria, and the development of primers for the amplification, in Pichia pastoris, by the Gibson Assembly method, of the genes of interest. The part BBa_K4496000 consists of a genetic code encoding the Jararhagin protein, responsible for causing hemorrhage in ophidic accidents, optimized for synthesis in Pichia pastoris yeast. Part BBa_K4496001 consists of a composite consisting of the parts of our 3A Assembly circuit for the bacterium Escherichia coli: the promoter BBa_J23100, the RBS BBa_J61101, the Jararhagin gene optimized for Escherichia coli, and the terminator BBa_B0015. The BBa_K4496002 part consists of a genetic code encoding the BJ46a protein, responsible for inhibiting the hemorrhagic protein Jararhagin, optimized for synthesis in Pichia pastoris yeast. The part BBa_K4496003 also consists of a composite consisting of the parts of our 3A Assembly circuit for Escherichia coli bacteria, but containing the gene encoding BJ46a optimized for Escherichia coli: the promoter BBa_J23100, the RBS BBa_J61101, the BJ46a gene optimized for Escherichia coli, and the terminator BBa_B0015. In addition to these parts, we have developed primers for performing protein expression and synthesis in Pichia pastoris yeast. The methodology to be used is the Gibson Assembly protocol and through this, it is necessary to use the developed primers during the amplification process of the genes.
The parts BBa_K4496004 and BBa_K4496005 are primers for the amplification of the Jararhagin gene upon expression in Pichia pastoris. The parts BBa_K4496006 and BBa_K4496007 are primers for amplification of the Jararhagin gene to insert it into the pPIC9K vector. The same was done for the BJ46a gene: parts BBa_K4496008 and BBa_K4496009 are primers for amplifying the gene for expression in Pichia pastoris; and parts BBa_K4496010 and BBa_K4496011 are primers for amplifying the gene to insert it into the pPIC9K vector. In addition, primers were developed for amplification of the psB1C3 plasmid (parts BBa_K4496012 and BBa_K4496013). Finally, primers (BBa_K4496014, BBa_K4496015, BBa_K4496016, BBa_K4496017) for both Jararhagin and BJ46a were made for expression in Escherichia coli bacteria.
Furthermore, other contributions consisted in the development of structural and kinetic modeling for the inhibitor. In structural modeling, in order to obtain a modeled protein closer to the real thing, the signal peptides were removed from the protein to then obtain the best possible spatial configuration. By means of bioinformatics tools it was possible to model the structure of the inhibitor BJ46a in order to obtain a structure that is faithful to the actual crystal of the protein. In addition, it is known that the inhibitor has glycosylations in specific regions, but these do not prevent or hinder the inhibition of Jararhagin (BASTOS, 2014). Thus, two circuits were developed, one for the synthesis of the protein by Pichia pastoris, possessing glycosylations, and one to try to produce in Escherichia coli bacteria, in order to innovate the biosynthetic route of production of the BJ46a inhibitor.
Kinetic modeling consists of developing a mathematical model that best describes the behavior of the protein in inhibitory assays. According to the literature, it is known that BJ46a inhibits the hemorrhagic protein in a non-covalent manner, i.e., reversibly, competing with the substrate for the docking site (BASTOS, 2014). Therefore, a whole mathematical work was developed to demonstrate, in a theoretical way, the expected predictabilities during inhibition assay experiments.
We have not only developed some structural models of our target proteins such as 𝛾PLI, PLA2, Bj46a and Jararhagin, but also have done some modeling regarding the enzymatic kinetics of the inhibitory effect of the interaction between inhibitor and enzyme, the crystal obtained should be useful for homology modeling for any iGEM team working on similar proteins, since snake venom proteins have high similarity between them in a genus. The data that would be obtained from the inhibitory assay would be useful to future researchers aiming to fully develop a synthetic snakebite serum, which will be great for the environment and will be one more step towards a animal testing free alternative for therapeutic drug testing. We also included some brand new parts in the Registry, which can be seen with more details here: http://parts.igem.org/Part:BBa_K4091024. Those parts are our target proteins and they should be useful to future iGEM teams and researchers aiming to enhance and further develop our idea.
Our parameters obtained in the experiments, as well as our methodology to find them can be useful to future iGEM teams, for many proteins and enzymes have not been fully characterized yet and our approach to this issue have been proven effective. To get our equation we adapted the Michaelis-Menten famous equation by solving the differential equation for time, giving us a function that shows the behavior of the concentration of a substrate A through the time in a batch reactor that can be applied in a real world scenario while developing the therapeutic substance. During our experiments we found the ideal temperature of melting of our primers, to do so we ran our primers on Thermo Fischer software (Olygoanalyser) to obtain a rough estimate of the temperatures of each primer in combination with our target genes. Then, we tested 5 temperatures using the estimate as the center point through the thermocycler. After a few attempts we managed to obtain the ideal conditions for amplifying our genes. Also, we strongly believe that this method is very useful for students and iGEM members that do not have access to high end softwares or precise estimates of the temperature of melting, during our experiments, we discovered that every gene is amplified at 62ºC. The exact range of temperatures may be found in the Notebook section, but we performed every PCR at 62ºC.
In addition, we performed enzymatic digestion of the genes from the PSB1C3 plasmid and used an adapted version of the 3A assembly method. Since our genes are relatively small (between 566 to 811bp), we digested the parts using the enzymes EcoRI and PstI separately, both at 37 degrees in the water bath for 2 hours, and between each step of this digestion we performed purification using the protocol Wizard SV Gel and PCR Clean Up System for best results. Although doing this process separately is more time consuming, we believe that it will enhance the 3A assembly process for some iGEM teams, for in many scenarios, the enzymes present in the mastermix will overlap each other during the reaction.