Team:ASTWS-China/Design

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Design

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In the beginning, our general idea is to construct an ideal tool for β-lactam detection and degradation. For the detective system, a sensitive and visualized biosensor with leakage-free devices may be a good choice. The lab-used E. coli provides an excellent reaction environment and enough materials for the biological pathway, and it is easy to dispose the engineered E. coli to avoid the spread of antibiotic-resistant bacteria in environment. Besides, it is necessary to reduce the β-lactams in water resources. As a result, we plan to design an antibiotic-degrading enzyme system to solve the problem. Details as follows.

Mec Detective System

We had read several research articles about β-lactam gene pathways, and finally our team utilized an efficient antibiotic-resistant gene pathway -- mec system in Staphylococcus aureus to redesign our detective system in E. coli. The mec system is comprised of a β-lactam sensor/signal transducer membrane protein mecR1, a gene repressor mecI and a mec operator. mecRI protein is a β -lactam sensor，whose surface domain binds covalently to the antibiotic. Binding of the β-lactams leads to the activation of cytoplasmic zinc-dependent protease domain of MecR1.The protease degrades the respective gene repressor MecI, which activates mec operator to regulate the transcription of the downstream gene. All the gene sequences were founded on KEGG database and NCBI database.

Figure 1 The mec pathway in Staphylococcus aureus (Blázquez, Blas, Llarrull L I, Luque-Ortega J R, et al., 2014)

Our detective system has 4 parts: mecR1, mecI, mec operator and mCherry. A mCherry (BBa_J06504), a red fluorescent protein, inspired by TU_Dresden 2017 iGEM team is added to the downstream of mec operator in order to make the biosensor more detectable. A certain content of β-lactams will change the structure of mecR1 so that it could degrade mecI repressor and release mec operator. As a result, The mec operator will activate the mCherry gene and cause mCherry to glow. We can see the E. coli turns red with naked eyes. Meanwhile, the β-lactam sensor mecR1 is under control of a strong constitutive promoter called Anderson Strong(BBa_J23100) while the mecI repressor is under control of a medium-strong constitutive promoter called Anderson Medium (BBa_J23110 ). Follow the mecI is the mec operator and its downstream mCherry. RBS(BBa_J34801) is added following each promoter.

Besides, we made “Anderson Strong-RBS-mecR1” and “Anderson Medium-RBS-mecI” in the opposite direction to ensure that the plasmid expressed two independent proteins as expected. We optimized the codon of mecI and removed the pstI cutting sites based on RFC10 cloning standard. All Biobricks were cloned into the pSB1C3 backbone and stored in DH5α.

Figure 2 Design of Mec detective plasmid.

CMY-10 Degraded System

The Enterobacter aerogenes β-lactamase blaCMY-10 (Gene Bank ID: AF357598)，which belongs to the group of ampC-related bla genes, is reported on a β-lactamase research article. It shows a strong ability to degrade β-lactams. Because of its excellent activity of β-lactams degradation, it is often used as a positive control in research. BlaCMY-10-pET24a is designed to put the blaCMY-10 gene in use. pET24a is an ideal protein prokaryotic expression vector for our research. It contains a cloning/expression region of the coding strand transcribed by T7 RNA polymerase and carries an optional C-terminal His Tag sequence. The protein expression is controlled by the lac operator, which is induced by IPTG, making it possible to control the procedure.

Figure 3 blaCMY-10 plasmid design

Detective System Plus-OxySP detection system

During our survey, we also found an interesting relationship between reactive oxygen species (ROS) and antibiotics. Antibiotics cause physiological changes associated with redox, which can lead to cell apoptosis. And different types of antibiotics generate varying levels of deleterious ROS, leading to cell death. The redox-related alterations triggered by bactericidal antibiotic stress are sensitive to antibiotics such as ampicillin, gentamicin and norfloxacin. Thus we decided to build another redox-related detective system to expand the scope of antibiotic detection. Three redox-relate promoters (called SoxSp, OxySp and KatGp) were found and SoxSp/KatGp were used in previous iGEM projects. So we planed to supplement the OxySp to this part collection and got its whole sequence from USE database EcoCyc (the bioinformatics database available at EcoCyc.org). The biosensor was designed based on the project of the 2018 Leiden iGEM team. The OxySp system has two parts: Oxysp promoter and mCherry. The existed antibiotics would activate OxySp promoter and stimulate the mCherry to glow, making it possible to see the E. coli turns red with naked eyes.

Figure 4 OxySp detection system design

References :

Blázquez, Blas, Llarrull L I, Luque-Ortega J R, et al. Regulation of the Expression of the β-Lactam Antibiotic-Resistance Determinants in Methicillin-Resistant, Staphylococcus aureus, (MRSA) [J]. Biochemistry, 2014, 53(10):1548-1550.

Llarrull L I , Mobashery S . Dissection of Events in the Resistance to β-Lactam Antibiotics Mediated by the Protein BlaR1 from\r, Staphylococcus aureus[J]. Biochemistry, 2012, 51(23):4642-4649.

Jeon J H , Kim S J , Lee H S , et al. Novel Metagenome-Derived Carboxylesterase That Hydrolyzes β-Lactam Antibiotics[J]. Applied and Environmental Microbiology, 2011, 77(21):7830-7836.

Structural basis for the extended substrate spectrum of CMY-10, a plasmid-encoded class C β-lactamase[J]. Molecular Microbiology, 2006, 60(4):907-916.

Lee S H , Jeong S H , Park, Y.‐M. Characterization of bla CMY-10, a novel, plasmid-encoded AmpC-type β-lactamase gene in a clinical isolate of Enterobacter aerogenes[J]. Journal of Applied Microbiology, 2010, 95(4):744-752.

Dwyer D J , Belenky P A , Yang J H , et al. Antibiotics induce redox-related physiological alterations as part of their lethality[J]. Proceedings of the National Academy of Sciences, 2014, 111(20):E2100-E2109.

Ranneh Y , Ali F , Akim A M , et al. Crosstalk between reactive oxygen species and pro-inflammatory markers in developing various chronic diseases: a review[J]. Applied Biological Chemistry, 2017, 60(3):327-338.

Seo S W, Kim D, Szubin R, et al. Genome-wide Reconstruction of OxyR and SoxRS Transcriptional Regulatory Networks under Oxidative Stress in Escherichia coli K-12 MG1655.[J]. Cell Reports, 2015, 12(8):1289-1299