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Quantitative detection and disposition system
Nanjing-China
Quantitatively detect and disposition system
Nanjing-China
Background
Physical and Chemical Methods
Fungal toxin TLC, ELISA, LC
Antibiotic LC, ELISA, CE, RIA
Heavy metal ion
ICP-AES/MS, AFS
Nature toxin ELISA, LC
Fungal toxin Heavy metal ion
Nature toxin Antibiotic
So dangerous! Traditional methods
Defects of these methods:
Complicated pretreatment
High cost
Professional manipulation
Weakness Other solution?
Background
Advantages of biological methods:
Environmental friendly
Energy saving
High sensitivity
Selectivity
Fungal toxin Heavy metal ion
Nature toxin Antibiotic
Other solution? Biological methods
Design
Plasmid
Plasmid
Sender of the detection part
Disposition and suicide system E.coli
Reporter of the detection part
Design
Results
Detection
Degradation
Biosafety
Sender
Generating AHL as signal molecules and create concentration gradient.
Reporter
Using quorum sensing to control the expression of RFP.
Design
Results
Detection
Degradation
Biosafety
Sender LuxI
LacI will be expressed when the promoter is activated by the target molecules.
AHL
LuxI will synthetize AHL.
Design
Results
Detection
Degradation
Reporter
Suicide
Design
Results
Degradation
Suicide
LuxR
LuxR is controlled by the constant promoter Pcons2, and is always expressed.
AHL
AHL is expressed by the promoter.
The combination of AHL and LuxR will starts the expression of LacI and CI.
CI
CI will stop the expression of the downstream LacI.
LacI
LacI can inhibit the expression of RFP.
Design
Results
Detection
Degradation
Suicide
Reporter
High concentration
The over-expression of LacI inhibits the expression of RFP.
Design
Results
Degradation
Suicide
Reporter
Medium concentration
CI can inhibit the expression of lacI, thus lacI can no longer Inhibit the expression of RFP.
Detection
Design
Results
Detection
Degradation
Suicide
Reporter
Low concentration
CI are too limited to inhibit the expression of lacI, thus the expression of RFP is inhibited by lacI.
Design
Results
Degradation
Suicide
Design
Results
Disposition
Suicide
LuxR LuxR is expressed by the constant promoter Pcons2.
LuxI Promoter recognizes the target molecules in the environment and begin to express LuxI.
AHL LuxI will promote the synthetics of AHL.
Positive feedback The combination of LuxR and AHL will start the expression of protein and LuxI.
Design
Next?
Results
Disposition
Suicide
What matters in the positive feedback system?
Design
Results
Degradation
Suicide
Without target and IPTG
When there is no target substance and IPTG, lysozyme will be expressed.
Design
How to prevent the bacteria from entering the environment?
Results
Degradation
Suicide
With target molecules
TetR will begin to generate when the specific promoter is activated by the target molecules, thus inhibiting the RFP.
Design
Design
Degradation
Suicide
With IPTG
IPTG activates Plac to start the expression of tetR, which can inhibit PtetO.
Results
Application
Application
Results
Does it work well?
We use heavy metal ion as an example to test it.
Au+
Application
Results DETECTION Modeling
Concentration: 0.01mol/L Diffusing time: 4000s Radius: 0.3cm
Au+
Application
Results DETECTION Experiment
Plasmid 1
Plasmid 2
Au+
Application
Results DETECTION Experiment
The plasmid 2 can inhibit the expression of RFP in plasmid 1.
Au+
Application
Results DETECTION Modeling
DISPOSITION
The luxI system and luxI and luxR system turned out to have the same result.
IPTG
Application
Results DETECTION Experiment
DISPOSITION
The system involves both luxI and luxR has the better result.
IPTG
Application
Results DISPOSITION DETECTION Modeling
SUICIDE
The lysozyme will increase with time without IPTG and target molecules.
Au+
Application
Results DETECTION Experiment
DISPOSITION SUICIDE
• 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.
Au+
Application
Results 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.
SUICIDE DISPOSITION
Application
Results
Problem 1: Anything else?
I will show you how many!
Application
Results
Library All the published promoters and enzymes can be used in our system.
Application
Results
Problem 2: How about those without specific promoter?
We can design riboswitches for them!
Application
Results
Application
DETECTION Riboswitch
We used RNAfold to simulate the secondary structure of the ribozyme, aptamers and riboswitches.
MC
Application
Results
Application
DETECTION Riboswitch
MC7 MC31
Modeling results are used to screen out the best two riboswitches.
MC
Application
Results
Application
DETECTION Sensor
Specific promoter for those we can find specific sensor
Riboswitch for those without promoters
Application DEGRADATION
DETECTION MlrD
The concentration of MC decreased faster with mlrD.
MC
Advantages Advantages Modularity
We can change the P and Protein with Promoter or riboswitch and functional protein in the library.
Advantages Human Practice
Positive feed back system
The positive feedback system can enlarge the signal of the target molecules .
Application
Any further application?
Polluted water
Cleaner water
Engineered bacteria
Human Practice Application
Team Communication Human Practice
Tsinghua-A Modeling and team management.
Jilin-China We provided mlrD to them.
NCTU-Formosa The 2nd iGEM conference.
Human Practice Publicity Efforts For people know little about iGEM
Society for students in other majors.
Invitation for the potential iGEMer.
Propagation for people unfamiliar with biology.
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
Acknowledgment Summary
Team
Practice&Policy
Team
Acknowledgment
