Team:TUST China/Detection
Design
We would like to thank all of those who have supported and assisted us in the laboratory, in the school and in society since the beginning of our tetracycline detecting and degradation project. Without those help, this project wouldn't have been possible and cannot been progressing well.
Overview
Detection Chasis
The first one is the which belong to the most important section of this double D system. Detecting the presence of tetracycline in the water sample is the first and most vital step among our project.
Through experimental discussion and design, we finally chose E. coli(DH5α) as our chassis organism to finish to constructed works. We need to detect tetracycline in water samples, so our chassis organism needs to be able to grow normally in the water samples containing tetracycline and complete our detection work. Therefore, we introduced gene tetA, gene tetR and gene tetO to achieve that the original E.coli can be obtained tetracycline resistance. At the same time, gene gfp is used as the visual output, therefore, it is more intuitive to judge whether tetracycline is contained in water samples. We successfully built the detection chasis which registered in the composite parts with its number:
BBa_K2685007
More than 40 genes encoding tetracycline resistance have been characterized to date and they are divided into 11 classes[1]. Among the various tet genes, the tet(A), tet(B), tet(D), tet(E) and tet(G) are reported in gram-negative bacteria. Comparing with those tet genes, we finally chose the tet(A) gene to construct in detection chasis to provide a tetracycline resistance[2][3]. tet(A) gene can code a transmembrane efflux protein that resistance to tetracycline. This is an energy-dependent process that decreases the accumulation of the antibiotic in whole cells. This protein functions as a metal-tetracycline/H+ antiporter.
The tetracycline repressor (TetR) is the repressor of the tetracycline resistance element; its N-terminal region forms a helix-turn-helix structure and binds DNA. Binding of tetracycline to TetR would reduces the repressor affinity for the tetracycline resistance gene (tetA) promoter operator(gene tetO) sites. And gene tetO is the tetracycline operon which can be binding the tetracycline repressor to control the expression of downstream genes.
In general, TetR protein binds to TetO domain to inhibit the expression of downstream genes. When tetracycline enters the E.coli, it will binds to free magnesium ions to form a complex and then acts on the TetR protein, making it unable to block the downstream pathway. Therefore, transmembrane protein TetA is expressed and excreted tetracycline chelates out of the membrane. At the same time, the GFP protein would also be coding to make the bacterial with green fluorescence.
We also want to increase the sensibility of the detectionn chasis, so we have exchanged the position of gene tet(R) and gfp. After this action, we have found that the bacterial would growth slowly compared with the nomal detection chasis.
References:
1. Møller, Thea S. B. et al. “Relation between tetR and tetA Expression in Tetracycline Resistant Escherichia coli.” BMC Microbiology 16 (2016): 39. PMC. Web. 16 Oct. 2018.
. Chopra, Ian, and Marilyn Roberts. “Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance.” Microbiology and Molecular Biology Reviews 65.2 (2001): 232–260. PMC. Web. 16 Oct. 2018.
3. Hedayatianfard, Keshvad, Mostafa Akhlaghi, and Hassan Sharifiyazdi. “Detection of Tetracycline Resistance Genes in Bacteria Isolated from Fish Farms Using Polymerase Chain Reaction.” Veterinary Research Forum 5.4 (2014): 269–275. Print.
