Difference between revisions of "Team:Shanghai Metro/Engineering"

Biological products produced by biotechnology, especially industrial microbial fermentation technology, have been widely used in food, medicine, animal feed and daily chemical products. With the development of strains and genetic modification technologies, the application range of industrial microbial strains will be further expanded, and its market scale will continue to grow. However, in recent years, intellectual property disputes related to industrial microbial strains have also been increasing year by year. According to the currently publicly reported cases, the main considerations for determining the intellectual property rights of industrial microorganisms include microbial morphology, DNA fingerprinting and similarity of conservative DNA sequences. Due to the wide distribution of microbes, the limited morphological characteristics that can be identified for individual microbes, and the strong conservativeness of some microbial genomes, the process of confirming and protecting related intellectual property rights is very expensive and lengthy. From the perspective of preventing the propagation and passage of strains, the development of concise and effective patented strain protection technology has become an urgent problem to be solved.

Type VI protein secretion system (T6SS) is a protein secretion system widely distributed in Gram-negative bacteria and can act on both eukaryotic cells and bacterial cells. Under natural conditions, bacterial cells encoding T6SS transfer cytotoxic or antibacterial effector factors (amidase, glycoside hydrolase, lipase, etc.) to recipient cells through physical contact, thereby inhibiting the growth of recipient cells. The bacteria that encode T6SS can translate and produce corresponding immune proteins to counteract the damage caused by toxic effectors.

The effector factors of T6SS were designed to constitutively express in industrial strains, and the corresponding immune proteins are designed in a controlled and induced mode (for example: arabinose, galactose, tetracycline, Zn2+). Only by adding specific inducers to the fermentation broth can the normal growth of industrial microbial strains be ensured. This can effectively prevent patented strains from being improperly stolen, and have concise and effective indicators for determining rights when disputes occur, thereby better protecting the intellectual property rights of strains from infringement.

Tke2 and tke4 are key functional factors that inhibit bacterial growth which are controlled tet promotor (Figure 1). The immunity effectors ike2/4 are under lac promotor. The anti-bacterial effectors tke2/4 and the immunity effectors ike2/4 were inserted in pUS232 vector (Figure 2).

pUS232-ike2 or pUS232-ike4 plasmids were transferred into bacterial strains (Escherichia coli DH5α). The expression of immune effectors needs to be induced by tetracycline in the appropriate concentration.

As seen in Figure 3, the control DH5α yield blu strains but the experimental group pUS232-ike4 had no blue strains, which could indicate that our design worked in DH5α. In addition, we also determined the concentration range of tetracycline for the experimental bacterial species growth by measuring the growth curves under tetracycline with different concentrations.

In order to determine the appropriate tetracycline induction concentration range for better growth of our patent strains, we tested the OD600 of the strains under various concentration of tetracycline and collected the data against hours and built the model. According to the scatter-plot analysis by MATLAB, the data points have a tendency of oscillation decline, and there is no model of elementary function conforming to this trend. Therefore, we decided to use interpolation method to study the numerical transformation and obtained the final curve.

Interpolation method selection: although the curve obtained by spline interpolation (Spline) is the smoothest, it will produce negative values, which does not conform to the actual situation. Therefore, Hermite interpolation method (Pchip) is chosen to keep the shape of the original data.

As the curve above, the X-coordinate shows the concentration of tetracycline and the Y-coordinate shows the corresponding OD₆₀₀ value of the strain D. In order to find the appropriate range of the tetracycline concentration, we referred the OD₆₀₀ value of the blank control strain after 6 hours without tetracycline:

Therefore, we drew a horizontal line where OD600 equals 0.3268 and find the cross points of these two lines where Xs are 17, 26, 78 and 113. As seen from the graph, it indicates that the growth of our patent strain (strain D) could grow normally even better the normal strain (strain E) when we add the tetracycline within the range from 17 µg/L to 26 µg/L, or the more recommended 78 µg/L to 113µg/L. To sum up, this model and this recommended concentration range could be used for the future application of our patented-strain protection technology.