Team:USP-EEL-Brazil/Experiments
Due to the pandemic situation, we only had access to the laboratories in September. Moreover, our genetic syntheses did not arrive in time. Thus, we did not have enough time to carry out the experiments of project Honorato. However, we intend to follow up on them in order to publish a scientific paper.
The experiments described on this page represent what we are going to do. They are the result of our research.
The microorganism chosen for the reproduction of 𝛾PLI, also called chassis, was the bacterium E. coli, selected because it is well characterized in the bibliography and presents easy culture control.
The 3A assembly is a technique for recombining two or more Biobricks, it uses restriction enzymes from the prefixes and suffixes of the Biobricks that will be cleaved at these points, and then recombined so that the prefix of one is recombined with the suffix of the other.
The advantage of this technique is that PCR and DNA electrophoresis are not necessary, and it has high success rates compared to conventional techniques.
In order for the bacteria to incorporate the plasmids they need to receive the right chemical signal and perform a thermal shock to the cells, by increasing the temperature, which makes their membranes more permeable to the entry of these molecules due to the pores that are formed by the pressure difference between the outside and inside of the cell.
The most widely used method for protein purification is using chromatography columns. Through characteristics of the protein, such as size, total charge, or affinity, it is possible to separate the enzyme of interest and other products generated in the culture.
The column contains a porous solid with chemical characteristics that match the type of protein to be purified. This is the stationary phase and is held in a buffered solution. In the mobile phase, the protein to be purified is diluted in this buffer, and migrates through the solid, occurring the separation between the protein of interest and other compounds (NELSON; COX, 2018).
The coding sequence of 𝛾PLI (EU155170.1) was identified through databases, such as NCBI (National Center for Biotechnology Information), KEGG (Kyoto Encyclopedia of Genes and Genomes).
It will be edited using software for signal peptide removal and codon optimization in order to optimize the chances of successful expression. To facilitate purification, a polyhistidine sequence will be added. The edited sequence will be sent to a company specialized in gene synthesis so that it can be used in the project.
For the production of the enzyme of interest a compound (BBa K525998) of a T7 promoter and RBS will be used, aiming at frequency of gene transcription, and for being a Biobrick well characterized by iGEM times.
A double terminator (BBa_B0015), formed by an E. coli terminator (BBa_B0010) and a T7 (BBa_B0012), widely used in E. coli, will be used to terminate the transcription, due to its synergy and efficiency, where the E. coli terminator also guarantees the end of transcription by the T7 RNA polymerase and E. coli polymerase.
Using the 3A Assembly technique, recombination of the gene sequences will be performed to fit them into the iGEM pattern and obtain the desired cassette.
Transformation techniques, such as heat shock, will be employed for insertion of the exogenous DNA into the E. coli BL21 chassis organism. To verify plasmid incorporation, the microorganisms will be selected using selective medium with antibiotic, in this way the resistant cells will be the ones that incorporate the 𝛾-PLI production plasmid.
Subsequently, the microorganisms will be selected using plain LB medium with chlorophenicol 30 mg L-1, then colony PCR will be performed and verification of the gene of interest by electrophoresis. The selected colonies will be grown in liquid LB medium for production of the protein of interest, and then go through a centrifugation process, causing the bacteria containing the enzyme to precipitate, forming pellets, which will be frozen at -80 °C.
As a next step, the pellets will be thawed, resuspended in lysis buffer, and the sonication technique will be applied to break the bacterial cell wall. At the end of the procedure, a supernatant containing the enzymes of interest will be collected and purified using an affinity chromatography column. In the last purification step, the samples that present absorbance peaks will be collected, and the presence of the enzyme of interest will be verified by electrophoresis and biochemical tests.
The standard mixture assay contains 200 µL of buffer (10 mM Tris/HCl, 10 mM CaCl2 and 10 0mM NaCl, pH 7.8), 20 µL of 4-nitro-3-octanoyloxy benzoic acid (4N3OBA) as substrate resulting in the final concentration of 0.25 mM, 20 µL of pure water and 20 µL of PLA2 (approximately 20 µg), will be incubated at 37 °C for 40 min. The hydrolysis values will be determined by absorbance at 425 nm at 5min intervals. The inhibitory effect of 𝛾PLI on PLA2 will be measured by changing absorbance following an incubation of PLA2 (20 µg) and 𝛾PLI (20, 10, 5 and 2.5 µg), mixed for 20min at 37 °C. All assays will be done in triplicate, and the absorbance at 405 nm will be measured using a spectrophotometer.
By means of the absorbance the speed of substrate consumption (nmol/min) will be determined. The percentage of inhibition will be defined by linear regression within the range of 20 to 30 min. The substrate consumption rate (degree of slope), shall be calculated using the formula ((VoPLA2-Vo𝛾PLI)/VoPLA2)100. (SERINO-SILVA et al., 2018).
We hope, upon successful completion of the project, that our studies can contribute to a research base aimed at the study of fatal case falls and limb loss from ophidic accidents, by making available a protocol on the production of recombinant 𝛾PLI, and assays of its in vitro efficacy for PLA2 inhibition. And, with a low cost of 𝛾PLI production.
As an application proposal, as a request within a medal requirement in the iGEM molds, we propose the distribution of ampoules containing the recombinant inhibitor in ambulances and also first aid stations, aiming at immediate application after the bite, even before identifying the snake.
We also aim to contribute to the beginning of a basic research aiming the production of a future totally recombinant drug (produced by bacteria), so that in the future we can stop using animals, like the horse, for the production of serum.
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CONCEIÇÃO SOBRINHO, Juliana. Inibidor de fosfolipase A2 tipo gama da serpente Bothrops atrox (atPLI): estudos on silico e in vitro de peptídeos sintéticos e análise de sistemas de expressão heteróloga do atPLI. 2018. 142f. Tese (Doutorado em Biologia Experimental) - Programa de Programa de Pós-Graduação em Biologia Experimental (PGBIOEXP), Fundação Universidade Federal de Rondônia (UNIR), Porto Velho, 2018.
SERINO-SILVA, Caroline. Purificação e caracterização do primeiro inibidor de fosfolipases A2 do tipo gama presente no soro da serpente Bothrops jararaca. 2017. 62 f. Tese (Doutorado) - Curso de Mestre em Biotecnologia, Universidade de São Paulo, São Paulo, 2017.
SANTOS-FILHO, Norival Alves. Caracterização Funcional e Estrutural de uma Fosfolipase A2 Ácida Tóxica Isolada da Peçonha de Bothrops moojeni. Universidade de São Paulo Faculdade de Ciências Farmacêuticas de Ribeirão Preto, São Paulo, 2009
MAGALHÃES, A. Regina. Caracterização de enzimas em peçonhas animais: identificação de fosfolipases do escorpião Hadrurus gerstchi e atividades enzimáticas da arraia Potamotrygon falkneri. 2017. 97 f. Tese (Doutorado) - Curso de Mestre em Biotecnologia, Universidade de Brasília, Brasília, 2017.
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REGISTRY OF STANDARD BIOLOGICAL PARTS (comp.). Assembly/3A Assembly. Available at: http://parts.igem.org/Help:Assembly/3A_Assembly. Access on: May 13, 2021