. Contribution .
1. Introduction
The HUBU-4-CHN team has made significant contributions to the field of synthetic biology in 2024, particularly in the engineering transformation of Escherichia coli Nissle 1917 (EcN) as a chassis cell, achieving breakthroughs. EcN, as a non-pathogenic and non-immunotoxic probiotic, is highly suitable for the treatment of human diseases. One of the project highlights of the team is the use of bacterial outer membrane vesicles (OMVs) produced by EcN as a drug delivery system. These OMVs can be designed with specific targeting, enabling precise delivery of drugs to the treatment areas required. Moreover, the targeting elements of OMVs can be adjusted according to different therapeutic environments and diseases, thereby expanding the scope of treatment.
In terms of gene-editing tools, the team employed the Cas9 RNP complex from the CRISPR-Cas9 system, which has shown tremendous potential in cancer therapy. To synthesize this RNP complex, the team developed a novel RNP self-assembly system, which is more rapid and efficient compared to other synthetic approaches, providing new possibilities for the future development of synthetic biology.
Additionally, the development of synthetic biology has also brought new therapeutic strategies, such as the use of polymer-locked fusion liposomes (Plofsome) to deliver siRNA or CRISPR-Cas9 RNP complexes, effectively crossing the blood-brain barrier and delivering to the cytoplasm of glioblastoma multiforme (GBM) cells, thereby inhibiting GBM growth and reducing resistance to chemotherapy drugs.
The application prospects of EcN as a chassis cell are broad, and researchers have developed a series of tools based on EcN, including the expansion of plasmid toolboxes, conjugation strategies for DNA transfer, quantification of protein expression capabilities, and the establishment of gene engineering mediated by natural integrases of EcN and in vitro cell-free protein synthesis (CFPS) systems, all of which provide a powerful platform and strategy for the field of synthetic biology.
Bacterial OMVs, as an emerging drug delivery system, have shown potential in various biomedical applications, including cancer therapy and the development of anti-infectious vaccines.
Overall, the works of the HUBU-4-CHN team not only promote the application of synthetic biology in the fields of medical and health but also provide new directions and tools for scientific research and technological development in this area.
2. Ethics and Safety
Ethics:Our team has been deeply involved in a series of activities and conferences related to safety and ethics, and has co-authored an ethics manual on synthetic biology with other iGEM teams, establishing an ethical framework for synthetic biology. This is aimed at promoting the healthy development of synthetic biology technology, ensuring that its application adheres to ethical principles. Our project research and practice are continuously improved in a direction that is safe, responsible, and deeply educational. At the same time, we explore ethical topics in synthetic biology. Our synthetic biology ethics manual not only contributes to the iGEM community and provides guidance for future teams and backgrounds, but also provides an ethical guidance framework for synthetic biology researchers, policymakers, ethical review bodies, and the general public in China and around the world. It guides the research and practice of synthetic biology towards a more responsible and sustainable direction.
Safety:The Escherichia coli Nissle 1917 (EcN) strain used in our project is a probiotic naturally present in the human gut and has been clinically applied in some countries. It has also attracted extensive attention in the research of medical applications worldwide. It has been frequently used in the treatment projects of iGEM over the years, hence we place great emphasis on the safety of its application. We have collaborated and exchanged with other iGEM teams using EcN as a chassis cell this year, and have jointly authored a white paper on EcN. The aim is to provide guidance and reference for the use of EcN in the future iGEM community and globally.
3. Part
We have created several basic parts and five composite parts. Each of these composite parts has its own advantages, some innovate on existing technology, and some use modular technology, facilitating the use of the iGEM community while also promoting technological innovation. Here is an example introduction to them:
The CRISPR/Cas system, which includes the CRISPR (Clustered regularly interspaced short palindromic repeats) array and CRISPR-associated protein (Cas). These two parts assemble to form an RNP complex with gene-editing activity, which has a wide range of applications in the field of bio-therapy. Currently, traditional Cas9 RNP is mainly prepared in vitro, which is formed by incubating Cas9 protein and sgRNA obtained by in vitro transcription or chemical synthesis. However, both in vitro transcription and chemical modification synthesis of sgRNA are too costly and time-consuming, which is not conducive to its application in clinical treatment.
In this project, we use a one-step method to prepare the RNP complex, using E. coli as a microbial factory for the expression of Cas9 protein and the transcription of sgRNA, and completing the "self-assembly" of the complex inside the cell. Compared with traditional methods, this method does not require additional preparation of crRNA, and can quickly and massively prepare inexpensive Cas9 RNP complexes through a one-step purification method. The RNP prepared by this method has extremely high stability and can maintain activity for more than a year under RNase-free conditions. This one-step method of preparing co-expression self-assembled RNP complexes is an important contribution to the future study of gene-editing complex targeting delivery platforms.
In addition, our team's project has produced a delivery platform for Cas9 RNP gene-editing complexes based on OMV. The designed targeting elements on the membrane surface can be modified in a modular manner, that is, by changing the specific antibodies connected to the proteins displayed on its surface, to achieve targeted gene editing of different cells. This modifiable modular part provides a composite part with strong operability for future iGEM teams.
