EHSBNUEthical and Ecological Considerations in Factor G Production
Our project focuses on creating a sustainable and humane method for producing Factor G, a key diagnostic component for deep-seated mycosis. The traditional method of extracting Factor G from horseshoe crabs harms their health and survival, threatening their populations and marine ecosystems. By using E. coli and Sf9 to produce Factor G, we offer a more sustainable alternative that preserves natural resources and reduces ecological impact.
Inspiration Behind the Sustainable Factor G Initiative
Inspired by the science fiction novel "Blue Blood Man," we became curious about blue blood on Earth. We discovered that Tachypleus tridentatus have blue blood with significant medicinal value, but widespread blood harvesting has endangered the species, especially during their breeding season. To preserve this resource and protect the species, we focused on developing an artificial alternative, which led to our project.
Objectives of the Factor G Optimization Project
Factor G, a heterodimeric serine protease zymogen, is activated by (1→3)-β-D-glucans (BDG) from fungal cell walls and is used in diagnostic agents for deep-seated mycosis. Extracting Factor G from horseshoe crabs is time-consuming and harms their health, leading to increased mortality after blood donation. Our goal is to produce Factor G using E. coli and optimize the protein structure to improve its binding with BDG. Ultimately, we aim to develop a more efficient version of Factor G and create an improved version of the TAL (Tachypleus Amebocyte Lysate) test.
Comprehensive Methodology for Sustainable Factor G Production
To achieve our project goals, we employ a systematic approach that encompasses a range of molecular biology techniques and innovative technologies. Initially, we constructed recombinant plasmids by synthesizing the Factor G sequence and agar gel electrophoresis for verification. We then utilize pET vectors, such as pET-28a(+) and pET-22b(+), with appropriate restriction enzymes to create compatible ends for cloning. The ligation reaction(in-fusion) fuses the vector and target gene, followed by transformation into E. coli strains like BL21(DE3) and Rosetta, with antibiotic screening to select for successful incorporation of the recombinant plasmids.
Expression and purification of Factor G are facilitated through culturing, IPTG induction, SDS-PAGE, Western Blotting, and nickel affinity chromatography. The BDG Assay System evaluates substrate decomposition, and the innovative Alpha Fold technology simulates the Factor G structure to identify structural variations. These variations are then incorporated using newly designed primers, and the cycle of expression, purification, and assay repeats to measure the impact of the modifications.
The optimal variant is selected for integration into a G-test kit, which is prototyped and compared with existing commercial models. This comprehensive methodology ensures a thorough investigation and optimization of Factor G, which leads to the development of a superior variant with enhanced properties.
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Image 1: Project methodology
How would the methods solve the problem:
Our systematic approach ensures a thorough investigation of the Factor G sequence, from its initial construction to the optimization of its expression and function. It ultimately leads to the generation of a superior variant with enhanced properties.
First, the construction of recombinant plasmids begins with synthesizing the Factor G sequence and injecting it into plasmids. The initial step is the reverse transcription of DNA, followed by PCR amplification, and the product is subsequently checked by using agar gel electrophoresis. Following this, a pET vector is constructed. A system with two plasmids is suggested due to the large size of each subunit, with the size of approximately 2000 base pairs. Appropriate pET vectors such as pET-28a(+), pET-22b(+) are selected, and restriction enzyme combinations like BamHI/HindIII and EcoRI/HindIII are used to treat the vector and create compatible ends. The site information is obtained by consulting the vector's multiple cloning site (MCS) sequence (restriction enzymes correlates to the sites) .
The next step is the ligation reaction. By in-fusion, the restricted vector and target gene fragments are mixed and DNA ligase is used to form a recombinant plasmid. The transformation of E. coli is then carried out, with the recombinant plasmid being introduced into host cells such as BL21(DE3) and Rosetta via chemical transformation. There are also Origami chemically competent cells in the system. Antibiotic screening, such as with kanamycin and ampicillin, is used to select bacterial cells containing both recombinant plasmids.
Then, the expression and purification of Factor G are the next goals. After culturing in LB or TB medium until adequate density, the expression of Factor G is induced under selected conditions, such as IPTG induction, and the expression level is detected using SDS-PAGE and Western Blotting. After successful expression, the target protein is purified using nickel affinity chromatography. Then, the BDG Assay System is employed to detect substrate decomposition. Under conditions where BDG and substrate are provided, the change in product concentration, specifically the amount of pNA, is measured using a microplate spectrophotometer.
The final stage of this process leverages the innovative Alpha Fold technology. Alpha Fold is used to simulate the structure of the Factor G amino acid sequence, and to identify possible structural variations. Based on these findings, new primers are designed to achieve these structural variations. The process then repeats with the reconstruction of expression systems, and the utilization of the BDG Assay System to measure the effect of the variant on substrate decomposition. The results are compared to those of the original Factor G to evaluate the improvements and effectiveness of the structural variations introduced through the Alpha Fold-guided modifications.
The optimal variation would be involved in the G-test kit, which will be later used to construct prototype kit and perform comparison tests with current commercial (animal based) models.
Transformative Impact of Sustainable Factor G Production
The development of rLAL-based fungal infection assay kits offers significant social, ethical, and policy benefits:
In the long run, rLAL-based assays will:
Ultimately, these advancements enhance public trust in healthcare and demonstrate leadership in ethical, sustainable practices globally.