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Team:SYSU-China
From 2012.igem.org
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Description
No matter in conventional genetic engineering or synthetic biology, we transfect the chassis bacteria, such as Escherichia coli and Bacillus thuringiensis, with the vector where the target gene is inserted and then get the engineering bacteria. But here comes two problems. The first one, when we use the engineered bacteria outside the laboratory that the horizontal gene transfer (HGT) of the extern gene between the engineered bacteria and other wild species may cause uncertain danger or even biohazard. And the second one, we have difficulty maintaining a high copy number of the vectors (which is also called Plasmid Addiction) between different generations and gaining the genetic stability of the engineered bacteria.And thus one of the targets of the project is to construct a new system of geneguard which will has two functions: 1. preventing the HGT between engineered bacteria and wild type, the HGT will cause the new host to die and insure the biosafety 2. maintaining a high copy number of the vectors in host bacteria, which can improved the stability of the engineered bacteria.
Background
Horizontal gene transfer (HGT): It refers to the transfer of genetic material that happens independently of reproduction. In bacteria, there are several mechanisms for HGT: transformation, transduction, bacterial conjugation, and gene transfer agents. The target of the geneguard is to prevent its happening.
Plasmid Addiction: In almost all microbe systems, the low copy number of the plasmids (or other vectors) may cause the loss of the target gene and the genetic instability of the engineered bacteria. The system of the plasmid addition contains a pair of stable toxin and an unstable antidote. When the plasmid is missing, the unstable antidote is decomposed by the enzyme, and the toxin causes the plasmid-missing bacteria to die.
Solution
- Find two proper pairs of toxin and antidote, named the Pair 1 and Pair 2.
- Plasmid Vector: Insert Toxin 1 gene and Antidote 2 gene in the Plasmid vector.
- Bacteria Genome: Insert Toxin 2 gene and Antidote 1 gene in the bacteria genome through transposon.
i. When HGT happens, due to the missing of the Antidote 1 gene and its production in the new host, the Toxin 1 will cause the new host to die.
ii. When the plasmid vector is missing, the missing of the Antidote 2 will cause the cell to die. And statistically, the intact host cells will be the overwhelming majority. And the Plasmid Addition is established.
- We choose the GFP and RFP as the reporter.
- We will choose as many pairs as we could and use mathematical modeling to model the system and optimize the efficiency of the different combinations.
Future work
After the construction of the new vector geneguard system, we could test it on a safer and more proper bacterium Wolbachia sp.(chassis geneguard system). We could standardize the experiment technique of Wolbachia and construct the new vector which is exclusive to Wolbachia. With the help of the genetic engineering of Wolbachia, we could improve the immunity of host of Wolbachia, then kill the pathogen before they could affected us.
Wolbachia: they are intracellular 伪-proteobacteria of the order Rickettsiales that infect insects as well as isopods, spiders, scorpions. They also have a remarkable feature that they have a reproductive abnormality which is called cytoplasmic incompatibility (CI). And CI causes the Wolbachia-infected ones to become the majority in their population, and this kind of species replace strategy make it possible to spread the engineered Wolbachia quickly in the infected area and block the spread of the disease, such as malaria, dengue and so on. And at the same time, the Wolbachia subspecies have a relatively strict limit in spread in different host species. All the advantages above give Wolbachia a great potential to become a chassis bacteria species.
