Team:Shanghai Metro/Engineering
Engineering
Background
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.
Design
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.
Build
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).
Figure 1. T6SS effector and immune protein expression box.
Figure 2. Schematic map of T6SS effector and immune expression plasmids.
Test
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.
Figure 3. Escherichia coli DH5α harboring pUS232-ike4 plasmid. Left: bacteria with pUS232 vector;
right: bacteria with pUS232-ike4 plasmid.
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.
Figure 4. Scatter-plot analysis by MATLAB.
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:
Table 2. OD600 value of the strain E: Pus232 after 6 hours
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.Regarding the range from 0 to 17 µg/L where we didn't have samples to verify the accuracy, thus we
didn’t count it as the recommended concentration range.
To sum up, this model and this recommended concentration range could be used for the future application of
our patented-strain protection technology.