Team:XHD-ShanDong-China/Implementation
Proposed Implementation
As an important model organism, E. coli plays an important role in current bioengineering. Since E. coli can produce human enzymes through recombinant DNA technology, it is widely used to produce useful compounds or pharmaceutical enzymes (such as human insulin, vaccines). The use of microorganisms to produce fuels and chemicals requires not only high production capacity of microorganisms, but also good environmental tolerance, such as high temperature resistance and acid and alkali resistance.
According to the experimental process designed by our project, degP-deficient strain MG1655_ΔdegP, degP wild-type strain with chromoprotein amilCP expression gene MG1655_LCP. And two experimental strains that changed the position of degP and upstream regulatory genes through gene editing: MG1655_LDC, MG1655_HDC. The experimental design of this project aims to screen out the heat-resistant strain MG1655_HDC by comparing the phenotypes of different strains. As heat-resistant E. coli chassis cells, it can be used in the following areas:
Figure 1. Schematic diagram of experimental design flow chart.
Using microorganisms to produce fuels and chemicals requires not only high production capacity of microorganisms, but also good environmental tolerance. For example, in view of the frequent temperature fluctuations in industrial production, it is necessary to select a microorganism with better thermal tolerance. At present, E. coli has shown good application prospects in the production of butanol, 1,4-butanediol, poly-3-hydroxybutyrate-4-hydroxybutyrate and other chemicals. Therefore, improving the thermal tolerance of E. coli has a certain value in promoting the application of E. coli in industrial production.
During the fermentation process, the temperature easily rises above the optimal temperature due to the strong metabolic activity of the cells. Therefore, we need to use a lot of water to cool the fermentation tank, otherwise the microorganisms will die or stop working. However, if we use this gene-edited bacteria that can survive at higher temperatures without compromising productivity, the cooling cost will be greatly reduced. In addition, since the genome of bacteria has been basically edited, heat resistance traits can be stably inherited, so it is very convenient in practical use. Enhancing the temperature tolerance of strains can reduce the cost and other problems caused by cooling in fermentation production, and ensure that industrial fermentation can be carried out normally at high temperatures. In addition, in the process of using biological fermentation to produce organic fertilizer, the activity of some enzymes will increase as the temperature rises, and heat-resistant strains can also be used in these areas.
In desert areas, in order to prevent sandstorms, there is a sticky protein that fixes the sand particles together. However, the high temperature environment in these tropical areas will make the engineered bacteria unable to work well. Therefore, if the heat-resistant engineered strains can be transformed, it will be suitable for tropical areas. The implementation of some of the projects has made very significant fundamental contributions.
In addition, many iGEM teams intend to use E. coli to solve environmental problems. However, some high-temperature regions, such as Ethiopia in Africa, may not be an ideal environment for microbial tools. In this case, our design to improve the thermal adaptability of E. coli by changing the distance between genes may provide an innovative solution.
Synthetic biology technology uses the concept of engineering design to design, modify and even re-synthesize organisms. It has spawned the "third biological revolution" following the discovery of DNA double helix structure and genome sequencing. It is expected to respond to humans. Facing major challenges in health, environment, energy, resources and other fields, it provides new solutions. It has been selected as one of the “Top Ten Scientific Progresses of the Year” by magazines such as Nature and is considered to be one of the major disruptive technologies affecting the future. Using synthetic biology technology, through the excavation of heat-resistant components, artificial design and optimization, integration and assembly, the heat resistance of the host cell can be changed, and its tolerance in high temperature environments can be improved.
We use gene editing methods to transform and screen out heat-resistant strains. Compared with traditional biological methods, it provides a new idea and direction for basic microbial research. At the same time, our modeling and analysis are for our research and analysis. The change of gene distance in the specific feedforward loop network motif of Escherichia coli provides a better theoretical basis for its effect on heat resistance.
Our work can create a new idea for the iGEM team in the future. They will carry out more complex operations based on our existing work and ideas to achieve their experimental goals. We hope that the future iGEM team can not only use the various components we create for its experimental operations, but also solve some problems from the perspective of gene space distance based on our ideas, thereby adding more vitality to the research in the field of synthetic biology.
However, for most of the production strains, due to their complex genetic background and incomplete understanding of the high temperature tolerance mechanism of E. coli, the new strains currently obtained are still far away from actual industrial production. At the same time, in the process of using gene editing technology to improve E. coli resistance, there are also the following problems that need to be resolved:
- 1. The range of substrates that can be applied to the selected breeding bacteria is relatively narrow and needs to be further expanded;
- 2. The high temperature tolerance of the selected breeding strain needs to be improved;
- 3. The genetic stability of related strains needs further evaluation;
- 4. The fermentation process (such as cell immobilization, simultaneous saccharification and fermentation, two-step fermentation, etc.) urgently needs optimization and innovation. In addition, the biosafety issues that may be caused by genetically modified strains require further evaluation and supervision.
- 5. Establishment of preservation methods and techniques for the selected heat-resistant strains
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