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Synthetic biology mainly explores and solves various biological problems by designing and assembling genetic elements with known functions and then transferring them into model microbial chassis cells. Its applications range from food, environment to medical treatment. However, the existing research methods still have some problems.

For example:

Artificially designed gene circuits are often disturbed by the endogenous complex gene network of the chassis microorganisms. How to reduce the interference so that the microbes can achieve specific functions according to our expectations is an important issue in the field of synthetic biology that needs to be resolved. Molecular biology, genomics, and minimal genome research are rapidly deepening our understanding of this complex function and minimizing this impact in engineering, but until now, our understanding is still very shallow.

At the same time, microbial cells have the characteristics of self-proliferation, which is often undesirable in many engineering applications. On the one hand, the growth and proliferation of microbial cells need to consume a lot of ATP, which reduces the energy flow obtained by functional circuits. On the other hand, the excessive proliferation of microbial cells is likely to cause biological pollution, which severely limits the application of genetically engineered microorganisms. What’s more, the various mutations that occur in the genetic process, will also bring troubles to research and application.


In order to address such problems , synthetic biologists try their best to find a balance between the complex and random biological system and the stable and predictable system required by engineering. The following studies are two examples, and they also give us a lot of inspiration.

(1) Minimum genome: It is the study of the minimum set of genes required by a certain organism. At present, there are a variety of efficient gene simplification methods and some lives that have undergone highly genetic simplification. But unfortunately, even if it is a life body regulated by only hundreds of genes, its complexity makes it impossible for us to complete the interpretation of the functions and relationships of all its genes[1].
From this, we learned that it is not feasible to control the work of organisms stably by analyzing the functions of the whole genome of life for the time being.

(2) Cell-free system: It is an in vitro system that uses mRNA or DNA as a template and supplements the enzyme system of cell extracts with various substrates and energy substances to synthesize proteins. Compared with intracellular expression, cell-free systems are more similar to chemical reactions, which we understand and control well. However, even the eukaryotic cell-free system cannot complete the modification of the high-level structure of the protein, and the complex cellular environment required for the expression of the product cannot be completely simulated[2][3].
From this research, we learned that for the functions of many organisms, the complete mechanism of cells and the complex cell environment are indispensable, that is, we can not simulate and reproduce complex cell functions through a “cell-free” system.

What are we going to do

Inspired by these studies, our project aims to overcome this complex effect by degrading the chromosomes of eukaryotic cells, and complete a series of applications with the help of the cell membranes, organelles, cell mechanisms and enzymes they still have. After experiments, we finally used the CRISPR system to target multiple copies of the yeast genome to achieve multiple chromosome breaks, successfully achieved chromosome degradation under the action of endogenous enzyme and obtained CREATE (Chromosome Released Eukaryote which is Active, Transitory And Environment-friendly). Subsequently, through multi-angle experiments, we verified the formation of CREATE, analyzed and purified it through flow cytometry.

We also designed experiments to verify the biological metabolic activity of chromosome-free eukaryotic cells CREATE, to test whether it can stably express artificial gene circuits as expected, and to explore the potential of chromosome-free eukaryotic cells as bioreactors. We also envisioned the potential of using the cell membrane of chromosome-free eukaryotic cells for membrane fusion as a delivery tool, as well as the application of using the fine structure of the cell membrane as a signal detector...

The importance of the project

The degradation of chromosomes means that the host cell's own complex gene network no longer exists, which may be able to relieve the cell's endogenous complex gene network from interfering with the exogenous artificial gene circuit. If the cell structure includes cell membranes, organelles and cell environment, as well as cell active substances such as enzymes, they can still exist for a period of time. During this time, we may be able to use the transferred artificial gene circuit to perform valuable work more stably.

At the same time, the degradation of the genome means that this special cell has lost the ability to grow and proliferate, reducing the possibility of accidental leakage of engineered organisms causing environmental pollution and damaging the ecological environment, and greatly improving the safety of the application of engineered organisms. This special property will make chromosome-free eukaryotic cells have more application possibilities.

We want to break through the limitations of the host genome’s complex effects on preset functions in engineering applications, so that strains can express artificial gene circuits more stably. At the same time, we hope to improve the safety of engineered strains modified by synthetic biology so that it can be used under safer conditions.


[1] Pelletier J F , Sun L , Wise K S , et al. Genetic requirements for cell division in a genomically minimal cell[J]. Cell, 2021(24 Pt A).
[2] Nakano H , Kawarasaki Y , Yamane T . Cell-free protein synthesis systems: increasing their performance and applications.[J]. Advances in Biochemical Engineering/biotechnology, 2004, 90(1):135.
[3] Kim T W , Oh I S , Ahn J H , et al. Cell-free synthesis and in situ isolation of recombinant proteins[J]. Protein Expression and Purification, 2006, 45(2):249-254.

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About Us

School of Chemical Engineering and Technology, Tianjin University, 135 Yaguan Road, Jinnan District, Tianjin