Difference between revisions of "Team:Shanghai Metro/Proof Of Concept"

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.

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).

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).

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.