Team:Shanghai Metro/Description

Shanghai_Metro

Description

Inspriation--Why we pick this topic for our project ?

An invention is a solution given to a technical problem that can be protected by a patent. Patents can control the commercial use of their inventions to protect the interests of inventors whose technology is truly groundbreaking and commercially successful. The inspiration of the project is based on the first patent dispute of microorganisms in China Regarding a pure white strain FINC-W-247 whose storage number was CCTCC No: M2012378. They prudently made a comprehensive judgment as infringement by comparing the important functional genes, regulators, important molecular markers, genes with higher differentiation ability and the sequences of ITS rDNA that are annotated by genome sequencing. However, there is still some controversy about those “evidence” like morphological contrast, sequences of ITS rDNA which shall be not enough to determine the identity. Even though, this case still took 2 more years to get closed. In another similar case in U.S, GlaxoSmithKline sought trade secret protection for its stolen strains. The company sued pharmaceutical giant Novartis and two of its subsidiaries in federal district court in Colorado for using a strain stolen from the plaintiff to produce a generic version of the antibiotic Augmentin. Many IPR disputes between pharmaceutical companies revolve around patent infringement. Due to the wide distribution of microorganisms, the limited morphological characteristics of individual microorganisms, and the conservative genomes of some microorganisms, intellectual property rights validation and protection are costly and lengthy. There is sill much insufficiency in the legal norm to deal with the patent dispute of microorganisms

Aiming to provide a fast, practical, and inexpensive way to protect patent rights over registered strains, our team designed to investigate a mechanism able to disable the strains if they’re stolen or replicated without authorization. This mechanism is developed based on the type IV secretion system (T6SS) of Gram-negative bacteria which would take effects on both eukaryotic cells and bacterial cells.

Solution - How we design the project?

Principle diagram of T6SS

Under natural conditions, bacterial cells encoding T6SS transport effectfactors with cytotoxic or antibacterial effects (amidase, glycoside hydrolyase, lipase, etc.) to recipient cells through physical contact, thus inhibiting the growth of recipient cells. The bacteria encoding T6SS can translate and produce corresponding immune proteins to counteract the damage caused by toxic effector factors.

With this idea coming from the T6SS, we planed to express the effector components of T6SS in industrial strains through inserting the gene segment tke2-ike2 to induce the controlled expression of the corresponding immune proteins. The normal growth of industrial microorganism can be ensured only by adding certain components in fermentation broth, so as to achieve the purpose of the experiment.

Work - what we did to achieve our goal?

In the summer of 2021, we came to the laboratory of Xuhui Central Hospital of Shanghai, China to conduct our experiments. We obtained TKE2 by PCR amplification according to the provided primer sequence. The plasmid Pus232 was then extracted from E. coli and linearized with SACII. A seamless cloning kit was used to clone TKE2 into linearized PUS232 to produce plasmid PUS232-TKE2. Pus232-TKE2 was digested with BamHI and XBAI double enzymes, linked with IKE2 PCR fragment which was also digested with enzyme, and transferred into Escherichia coli to obtain plasmid Pus232-TKE2-IKE2. In the biochemical section, the growth of strains containing plasmids and the blank group was measured. After the constructed plasmids entered the host bacteria, the growth of the transformed group and the control group in liquid medium (with/without tetracycline) would be measured by spectrophotometry (OD600nm) and gradient dilution plate method, respectively in order to prove our design.

The discovery and protection of bacteria have always been the focus of scientific protection, and the patented strain protection technology developed by us can be used for these companies that produce and keep patent strains.

Reference:

[1] 陈剑山, 郑服丛. ITS序列分析在真菌分类鉴定中的应用[J]. 安徽农业科学, 2007(13):25-26+32.

[2] Yanliang, F. (2005). Strain Protection Essentials. China Academic Journal, 54-51.

[3] Hernandez, R. E., Gallegos‐Monterrosa, R., & Coulthurst, S. J. (2020). Type VI secretion system effector proteins: Effective weapons for bacterial competitiveness. Cellular Microbiology, 22(9). https://doi.org/10.1111/cmi.13241

[4] Silverman, J. M., Brunet, Y. R., Cascales, E., & Mougous, J. D. (2012). Structure and regulation of the type VI secretion system. Annual review of microbiology, 66, 453–472.

[5] Ruzhen LI, Xiao-ben SHI, Jian-liang YU, Hong-hao YUE (2017). Bacterial Patent Analysis of Microbial Fermentation Feed. Contemporary Chemical Industry, 2101-2103

[6] Bingle, l.E.H. et al. (2008). Type VI secretion: a beginner’s guide. Current opinion in microbiology. 11:3-8.

[7] Hernandez, R. E. et al. (2020). Type VI secretion system effector protein: Effective weapons for bacterial competitiveness. Cellular microbiology. 22:e13241.