Team:Shanghai Metro/Proof Of Concept
PROOF OF CONCEPT
1. Theory and analysis
1) Rationale
The topic was inspired by the Type VI secretion system (T6SS) of Pseudomonas
putida. T6SS kill eukaryotic predators or its competitors by injecting anti-bacterial effectors, the toxic
substances, into eukaryotic or prokaryotic cells. Pseudomonas putida itself will not be killed by the
anti-bacterial effectors because it secrets immunity proteins specific to each of their antibacterial
effector proteins to protect itself. [1] Based on this theory, we want to transfer tke2/4, the
anti-bacterial effectors, and ike2/4, the immunity proteins to the plasmid of protected bacterial species.
Protect the bacterial species by controlling the immunity proteins.
2) Theory explanation
The DNA sequences of immunity protein ike2/4 and the anti-bacterial effector tke2/4
in Pseudomonas putida are extracted, and they are connected to Pus232 backbone by T4 DNA
ligase.[2] Then,
Pus232-ike2/4 is linearized by single enzyme digestion. Later on, the tke2/4 is connected to Pus232-ike2/4
through homologous recombination enzyme.[3] After employing a complicated process,
Pus232-ike2/4-tke2/4
plasmid is finally obtained. Tetracycline is then added to LB medium (solid) to induce the expression of
ike2/4, so that the bacterial species can survive eventually.
2. Product performance
In order to make sure that our protection plasmid works, we tested to find the
optimal amount of our inducer tetracycline and the growth speed of ike2/tke2 or ike4/tke4 types by using
OD600 testing.
Tetracycline amount determination:
● In order to eventually
determine the specific
tetracycline amount and make sure our protection plasmid works, we set up a control group with only Pus232
strains and a test group with Pus232-tke4-ike4 strains. We each added either none, 25µg/L, 50µg/L, 75µg/L,
100µg/L, 200µg/L, 300µg/L, 400µg/L, 500µg/L, 600µg/L, 700µg/L, 800µg/L, 900µg/L, 1000µg/L, or 2000µg/L of
tetracycline to the control group and test group. The test group results showed that the growth greatly
increased and possibly at its best with adding tetracycline concentrations at the range of
300-500µg/L(Fig.1), while the effect to Pus232 strain alone is less obvious(Fig.2). The control group
ensured that tetracycline alone harms growth of ordinary strains by showing a decreasing pattern of biomass
concentration adding tetracycline concentrations over 500µg/L(Fig.2).
Figure 1. Growth testing of test group Pus232-ike4-tke4 strains under different concentrations of
tetracycline.
Figure 2. Growth testing of control group Pus232 strains under different concentrations of
tetracycline.
3. Manual for users
Figure 3. Workflow of the product
When the patented strain holder receives our product, the protection plasmid can be
used for transformation directly into their patented strain, then select the positive colonies on the LB
plate with kanamycin. Pick several mono-colonies from the plate to culture as brand-new patented strains.
Tetracycline at the range of 300-500µg/L is required in the course of fermentation. If someone steals the
brand-new patented strains, they could not culture the strains or do fermentation because they do not know
the existence or a feasible concentration of tetracycline. Therefore, patented strain holder can protect
their patent(Fig.3).
Bibliography
1. Hernandez, Ruth E., et al. “Type Vi Secretion System Effector Proteins:
Effective Weapons for Bacterial Competitiveness.” Cellular Microbiology, vol. 22, no. 9, 2020,
doi:10.1111/cmi.13241.
2. Rossi, R. “Functional Characterization of the T4 DNA Ligase: A New Insight into
the Mechanism of Action.” Nucleic Acids Research, vol. 25, no. 11, 1997, pp. 2106–2113.,
doi:10.1093/nar/25.11.2106.
3. “Homologous Recombination in Procaryotes.” Microbiological Reviews, vol. 52, no.
2, 1988, pp. 304–304., doi:10.1128/mr.52.2.304-304.1988.