Team:NEU China B/Design
Design
Our inspiration came from the industrial fermentation. Among of fermentation bacteria Lactobacillus is the most well-known, which is mainly used in manufacturing yogurt and its derivatives. In modern life, yogurthas been considered as the indispensable food for your daily life.
Speaking of the yogurt, there is an ancient story said that nomads living in the Anatolian (now Turkish) plateau had made and drank yogurt as early as 3000 B.C. The original yogurt may be made by chance. Goat milk often spoiled in stocks since the bacteria contaminated the milk. However, in one case, air born lactic bacteria accidentally entered into the milk and lead to the milk deterioration. Unexpectedly, the “deteriorated milk” became more sour and sweet palatable, this is the earliest yogurt. Shepherds found the yogurt so good to drink that they inoculated the sample into the uncontamintated milk in order to retrieving the yogurt. After a period of culture and fermentation, a new yogurt was obtained.
Although fermentor is a great place for microorganisms rapidly grow, reproduce, and accumulate metabolized organic matter, it also could generate many by-products.. The excess lactic acid not only inhibited the growth of fermentation bacteria, but also reduced the quality of the product. Additionally, high concentration of industrial lactic acid may corrode human skin and epithelium. So we came up with the idea of making a real-time biophysical receptor to measure in real time whether the culture fluid in the fermentor need to be refilled.
Therefore, we designed a lactic acid monitoring circuit. By constructing a biophysical loop with the second Quorum Sensing system of Escherichia coli K12 strain, we would be able to use optical fiber probe to detect green fluorescence intensity.
We have known from reference [1] that the second Quorum Sensing system of Escherichia coli k12 strain takes DPD (45-dihydroxy-2, 3-pentanedione), which is expressed by LuxS Gene, as its signal molecule. The DPD can bind to the repressor protein LsrR after its methylation and phosphorylation. The repressor protein loses its function, hence the Quorum Sensing system activates. We integrated the GFP gene behind the gene LsrA\B\C\D\F\G (6x manipulation sequence) which operates the Quorum Sensing( Fig.2), for the purpose of detecting the intensity of fluorescence to determine the concentration of lactic acid in the environment.
According to the reference [2], we can learn that the O1-O2 site (fig.3) of LLDPR Operon is where the lactic acid spot and integrate. We jointed the LuxS Gene to the O1-O2 site and integrated it into the vector( pCFDuet plasimid). We attempted to obtain a large amount of LuxS gene expression under the suitable alarm moleccule concentration, and then started a simulated group effect system to express the green fluorescent protein (GFP).
The optical fiber detector is constructed by the students of the Information Institution.
Design
Design is the first step in the design-build-test cycle in engineering and synthetic biology. Use this page to describe the process that you used in the design of your parts. You should clearly explain the engineering principles used to design your project.
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What should this page contain?
- Explanation of the engineering principles your team used in your design
- Discussion of the design iterations your team went through
- Experimental plan to test your designs