Without chromosomes, the synthetic cells won 't proliferate freely or cause genetic contamination. Besides, interference or rejection with the genome of receptor cells and synthetic gene circuits will not happen. Although these cells will die ultimately, the enzymes can still remain active for a period of time, so these chromosome-free eukaryotic cells can be used in many aspects. Specifically, we can expect that in the foreseeable future, chromosome-free cells will stimulate the development of biomanufacturing, healthcare, agriculture, and environment fields.
CREATE, the chromosome-free cell, is a special reaction chassis. It has no chromosomes. Therefore, it doesn’t undergo genetic mutations that cause artificial genetic circuits to fail, and can’t proliferate so that it has good biosecurity. Besides, the biological enzymes in its body are active for a certain period of time, so that it can maintain the cellular mechanism for a period of time and respond to the artificially constructed gene circuit. This feature gives CREATE a variety of application possibilities. Our team has done a lot of HP work to assist in the design and idea modification of the application of CREATE. In the end, we chose to try in the direction of "using CREATE for drug production and delivery".
1.The idea of new type of microbial application ointment
Bacteria and fungi on wounds are very distressing. Ordinary bacteria and fungi are used as drug carriers, so they will multiply on the wound, which may cause serious consequence in the end. But CREATE as chromosome-free cells may change this situation. We decide to use chromosome-free cells to produce a protein that has a clotting effect and make it into a paste. When we apply this kind of ointment containing CREATE to the wound, it will continue to express coagulation protein to help the wound heal.
2.The idea of using CREATE to produce toxic chemicals
Eristostatin is a special biologically active protein with excellent medical effects in clinical treatment. It has a very large demand, but it is difficult to produce and purify. Because most of the eristostatin has antibacterial effect, it is toxic to cells. Expressing the gene of eristostatin in ordinary cells through genetic engineering may cause apoptosis, then it may leads to fail to obtain the expression product.
Eristostatin is a protein that can only be produced by eukaryotes. Yeast, as a commonly used eukaryotic engineering bacteria, is capable of processing eristostatin. At the same time, because of the loss of chromosomes, the toxic protein cannot make it "suicide". Therefore, using CREATE to produce eristostatin may be a feasible way for people to obtain this special protein in the future.
(1) Integrate the gene expressing eristostatin into the plasmid and transfer it into Saccharomyces cerevisiae cells with cleavage elements.
(2) Enrich the cell, induce the cutting element, and use the CRISPR system to cut the chromosome to construct CREATE.
(3) Induce the expression of eristostatin and use CREATE to produce the drug.
(4) Purify and package the expressed eristostatin.
Industrial production requirements for CREATE
|Easy to purify and separate||We will design a simpler way to separate CREATE, so that the preparation of CREATE can be cheaper, the steps can be simpler, and a large number of CREATE cells can be obtained in a short time. This will be our next job.|
|Lifetime||In CREATE cells, artificial gene circuits such as glycolysis pathways need to be constructed to supplement CREATE cells with the ATP and NADH needed for survival, so as to extend the life of CREATE as much as possible.|
|Maximum yield||We will try to regulate the flow of energy in CREATE, so that more energy and materials will be used to produce the materials we need. In a cell that has lost the ability to regulate chromosomes, it should be easier to achieve this than in a cell that still has chromosomes.|
Other fields where CREATE can be used
How to transmitting large DNA fragments has been bothered scientists for years. CREATEs lost their chromosomes can be used as an biologically active box, splicing DNA fragments and transmitting them. In this case, we can use chromosome-free eukaryotic cells as biological tools for transmitting large DNA fragments.
Unlike protocells that are liposomes or vesicles, CREATE retains its original bacterial cell membrane. The bacterial membrane houses many vital functions: sensing of environmental signals, generation of ATP via proton gradients created by the electron transport chain, the import of nutrients into the cell, and the export of waste out of the cell. Hence, CREATE not only have vast potential in elucidating the minimal requirements for life but can also be utilized as biocatalysts, biosensors, and more in sensitive environments.
Fan C , Davison P A , Habgood R , et al. Chromosome-free bacterial cells are safe and programmable platforms for synthetic biology[J]. Proceedings of the National Academy of Sciences, 2020, 117(12):201918859.