Quantitative detection and disposition system Nanjing-China
Quantitatively detect and disposition system Nanjing-China
Background Traditional methods Other solution? So dangerous! Weakness Defects of these methods: Physical and Chemical Methods Fungal toxin TLC, ELISA, LC Complicated pretreatment Fungal toxin Heavy metal ion Antibiotic LC, ELISA, CE, RIA High cost Heavy metal ICP-AES/MS, AFS Professional manipulation ion Nature toxin ELISA, LC Antibiotic Nature toxin
Background Other solution? Biological methods Heavy metal ion Fungal toxin Advantages of biological methods: Environmental friendly Energy saving High sensitivity Selectivity Nature toxin Antibiotic
Design
Design Sender of the detection part Reporter of the detection part Plasmid Plasmid Disposition and suicide system E.coli
Design Sender Generating AHL as signal molecules and create concentration gradient. Reporter tion Using quorum sensing to control the expression of RFP.
Design Sender Detection LuxI AHL LacI will be expressed when the LuxI will synthetize AHL. promoter is activated by the tion target molecules.
Design Detection Reporter tion
Design LacI LuxR CI AHL LuxR is controlled by the constant LacI can inhibit the expression of CI will stop the expression of the AHL is expressed by the promoter. promoter Pcons2, and is always RFP. downstream LacI. tion The combination of AHL and expressed. LuxR will starts the expression of LacI and CI.
Design Detection Reporter High concentration tion The over-expression of LacI inhibits the expression of RFP.
Design Reporter Detection Medium concentration tion CI can inhibit the expression of lacI, thus lacI can no longer Inhibit the expression of RFP.
Design Detection Reporter Low concentration tion CI are too limited to inhibit the expression of lacI, thus the expression of RFP is inhibited by lacI.
Design tion
Design LuxI AHL Positive feedback LuxR Promoter recognizes the target molecules in the The combination of LuxR and AHL will start the environment and begin to express LuxI. expression of protein and LuxI. LuxI will promote the synthetics of AHL. LuxR is expressed by the constant promoter Pcons2. Next? tion
Design What matters in the positive feedback system? Disposition
Design Without target and IPTG How to prevent the bacteria from tion entering the environment? When there is no target substance and IPTG, lysozyme will be expressed.
Design With target molecules tion TetR will begin to generate when the specific promoter is activated by the Suicide target molecules, thus inhibiting the RFP.
Design With IPTG tion IPTG activates Plac to start the expression of tetR, which can inhibit Suicide PtetO.
Does it work well? We use heavy metal ion as an example to test it. Au +
Au + Modeling DETECTION Concentration: 0.01mol/L Diffusing time: 4000s Radius: 0.3cm
Au + Experiment DETECTION Plasmid 1 Plasmid 2
Au + Experiment DETECTION The plasmid 2 can inhibit the expression of RFP in plasmid 1.
IPTG DISPOSITION Modeling DETECTION The luxI system and luxI and luxR system turned out to have the same result.
IPTG DISPOSITION Experiment DETECTION The system involves both luxI and luxR has the better result.
SUICIDE Au + DISPOSITION Modeling DETECTION The lysozyme will increase with time without IPTG and target molecules.
SUICIDE Au + DISPOSITION Experiment DETECTION • RFP is used in stead of lysozyme. • Au + and IPTG can prevent bacterium from suicide. • Engineered bacterium will die without Au + and IPTG in the environment.
SUICIDE DISPOSITION DETECTION The results mentioned above indicate that: AHL can form a concentration gradient and its concentration will decide whether RFP will be expressed or not. The positive feedback system is most effective when both LuxI and LuxR are replenished. Lysozyme will be expressed when there is neither target substance nor IPTG, thus killing the engineered bacteria.
Problem 1: Anything else? I will show you how many!
Library All the published promoters and enzymes can be used in our system.
Problem 2: How about those without specific promoter? We can design riboswitches for them!
MC Riboswitch DETECTION We used RNAfold to simulate the secondary structure of the ribozyme, aptamers and riboswitches.
MC Riboswitch DETECTION Modeling results are used to screen out the best two riboswitches. MC3 C31 MC7 C7
Sensor DETECTION Specific promoter for those we can find specific sensor Riboswitch for those without promoters
DEGRADATION MC MlrD DETECTION The concentration of MC decreased faster with mlrD.
ges Advantages Modularity We can change the P and Protein with Promoter or riboswitch and functional protein in the library.
Advantages Positive feed back system The positive feedback system can enlarge the signal of the target molecules . Any further application?
Application Polluted water Engineered bacteria Cleaner water
Team Communication Human Practice NCTU-Formosa Tsinghua-A The 2 nd iGEM conference. Modeling and team management. Jilin-China We provided mlrD to them.
Human Practice Publicity Efforts For people know little about iGEM Propagation Society for people unfamiliar with for students in other majors. biology. Invitation for the potential iGEMer.
Summary Constructed and tested 3 systems (detection & disposition & suicide) Submitted 20 parts and registered 50 parts (BBa_K1520506, BBa_K1520500,BBaK1520507) Consider biosafety from two aspects Helped other teams with their projects Introduced iGEM to laymen
Team Practice&Policy
Team
Acknowledgment
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