COMBINING EXPERIMENT,MODELING AND HUMAN PRACTICE TO PRODUCE: Our project combines molecular modeling, Human Practices, and market analysis to optimize LACS1 for sustainable DHA-PC production while addressing societal needs.
Key Points:
- MOLECULAR MODELING: Enhanced LACS1 efficiency using molecular docking, dynamics simulations, and flux balance analysis in Schizochytrium limacinum.
- HUMAN PRACTICE: Engaged in market surveys and educational outreach to understand consumer preferences and promote synthetic biology.
- SUSTAINABLE PRODOCUTION: Focused on biological synthesis of DHA-PC to meet market demands and promote environmental sustainability.
Contribution of the Experiment
Our project contributes significantly to the iGEM community by providing a novel approach to the biological synthesis of DHA-PC through the optimization of the LACS enzyme. Specifically, we engineered LACS to enhance its affinity for DHA using protein modeling and mutagenesis, which can serve as a foundation for teams focusing on fatty acid metabolism and biosynthesis. Additionally, the methodologies we developed for screening enzyme variants, coupled with advanced product detection techniques like LC-MS, offer robust tools for future teams working on metabolic engineering. Our work also highlights the potential of using *Schizochytrium limacinum* and *Saccharomyces cerevisiae* as production chassis, paving the way for sustainable production of complex lipids. This contribution addresses challenges in synthetic biology related to enzyme specificity and scalable biosynthesis, providing practical insights and resources for the iGEM community to further explore lipid engineering and DHA-PC production.
| Code | Name | Length | Description |
|---|---|---|---|
| BBa_K5255000 | Lacs1+his | 1980 | For more than 24 carbon has a certain catalytic capacity, including DHA |
| BBa_K5255001 | Czlacs5+his | 2145 | The activity of [C16:1] [C18:3n3] [EPA] [DHA] [C18:1] was significantly decreased from left to right |
| BBa_K5255002 | LACS6+his | 2763 | It has certain catalytic activity for DHA |
| BBa_K5255003 | MLACS1 | 1982 | Mutant Lacs1 has higher catalytic activity on DHA |
| BBa_K5255004 | pAR-Ec633 | 7602 | A nuclear plasmid encoding an error-prone mutant TP-DNAP1 (L477V, L640Y, I777K, W814N) for OrthoRep |
| BBa_K5255005 | FAS1 | 379 | It has been used to knockout the FAS1 gene of INVSC1, including a sequence of tRNA, HDV ribozyme, FAS1 gRNA, gRNA scaffold, and SNR52 terminator. |
| BBa_K5255006 | FAS1 FAA4 | 547 | It has been used to knockout the FAS1 and FAA4 genes of INVSC1, including a sequence of tRNA, HDV ribozyme, FAS1 gRNA, FAA4 gRNA, gRNA scaffold, and SNR52 terminator. |
| BBa_K5255007 | FAS1 FAA4 FAA1 | 715 | It has been used to knockout the FAS1, FAA4, and FAA1 genes of INVSC1, including a sequence of tRNA, HDV ribozyme, FAS1 gRNA, FAA4 gRNA, FAA1 gRNA, gRNA scaffold, and SNR52 terminator. |
| BBa_K5255008 | FAS1 homologous repair fragment | 182 | It is used to repair the FAS1 gene knocked out by CRISPR and facilitate PCR screening |
| BBa_K5255009 | FAA1 homologous repair fragment | 182 | It is used to repair the FAA1 gene knocked out by CRISPR and facilitate PCR screening |
| BBa_K5255010 | FAA4 homologous repair fragment | 182 | It is used to repair the FAA4 gene knocked out by CRISPR and facilitate PCR screening |
| BBa_K5255011 | pGKL1-LACS1 | 7051 | Linearized plasmid for homologous recombination with linearized pccl-LACS1 |
| BBa_K5255012 | pGKL1-Czlacs5 | 7216 | Linearized plasmid for homologous recombination with linearized pccl-Czlacs5 |
| BBa_K5255013 | pGRKL1-LACS6 | 7726 | Linearized plasmid for homologous recombination with linearized pccl-LACS6 |
| BBa_K5255015 | gal+LACS1+his | 2326 | LACS1-his fusion protein regulated by galactose promoter |
| BBa_K5255016 | gal+Czlacs5+his | 2491 | Czlacs5-his fusion protein regulated by galactose promoter |
| BBa_K5255017 | gal+LACS6+his | 8917 | LACS6-his fusion protein regulated by galactose promoter |
| BBa_K5255018 | T7+LACS1+his | 2005 | LACS1-his fusion protein regulated by IPTG |
| BBa_K5255019 | T7+Czlacs5+his | 2170 | Czlacs5-his fusion protein regulated by IPTG |
| BBa_K5255020 | T7+LACS6+his | 2680 | LACS6-his fusion protein regulated by IPTG |
| BBa_K5255021 | T7+MLACS1+his | 2007 | MLACS1-his fusion protein regulated by IPTG |
Contribution of the Modeling
Our project makes a significant contribution to the iGEM community by utilizing advanced modeling techniques to enhance the functionality of the LACS1 enzyme. We performed molecular docking and molecular dynamics simulations to identify optimal mutation sites, allowing for precise modifications to improve the enzyme's catalytic efficiency. Additionally, we conducted flux balance analysis of fatty acid metabolism within *Schizochytrium limacinum* , providing valuable insights into metabolic pathways and their regulation. This comprehensive approach not only demonstrates the potential of LACS1 for DHA-PC biosynthesis but also offers a methodological framework that other iGEM teams can adopt for enzyme engineering and metabolic modeling. Our findings contribute to the broader field of synthetic biology by facilitating the sustainable production of valuable fatty acids and lipids through enhanced biotechnological processes.
Contribution of Human Practice
Our project significantly contributes to the iGEM community by integrating comprehensive Human Practices, which included market resea # Contribution of Human Practice
Our project significantly contributes to the iGEM community by integrating comprehensive Human Practices, which included market research, educational outreach in schools, and public awareness initiatives. Through market surveys, we gathered valuable insights into consumer perceptions and preferences regarding DHA-PC, ensuring that our biological synthesis approach aligns with market needs. Our educational efforts in schools aimed to raise awareness of synthetic biology and its applications, fostering interest in biotechnology among the next generation. Additionally, our public education campaigns provided accessible information about the benefits of sustainable bioproduction and the importance of natural products, thus promoting informed discussions within the community. By addressing societal concerns and enhancing public understanding, our Human Practices initiatives not only support the success of our project but also contribute to the overall acceptance and integration of synthetic biology solutions in everyday life, paving the way for future iGEM teams to engage with their communities effectively.
