disposition system Nanjing-China Quantitatively detect and - - PowerPoint PPT Presentation

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

tion

Sender

Generating AHL as signal molecules and create concentration gradient.

Reporter

Using quorum sensing to control the expression of RFP.

Design

Detection tion Sender

LuxI

LacI will be expressed when the promoter is activated by the target molecules.

AHL

LuxI will synthetize AHL.

Design

Detection tion Reporter

Design

tion

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

Detection tion Reporter

High concentration

The over-expression of LacI inhibits the expression of RFP.

Design

tion Reporter

Medium concentration

CI can inhibit the expression of lacI, thus lacI can no longer Inhibit the expression of RFP. Detection

Design

Detection tion Reporter

Low concentration

CI are too limited to inhibit the expression of lacI, thus the expression

• f RFP is inhibited by

lacI.

Design

tion

Design

tion

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?

Disposition

What matters in the positive feedback system?

Design

tion

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?

tion 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

tion Suicide

With IPTG

IPTG activates Plac to start the expression of tetR, which can inhibit PtetO.

Does it work well?

We use heavy metal ion as an example to test it.

Au+

DETECTION Modeling

Concentration: 0.01mol/L Diffusing time: 4000s Radius: 0.3cm

Au+

DETECTION Experiment

Plasmid 1 Plasmid 2

Au+

DETECTION Experiment

The plasmid 2 can inhibit the expression of RFP in plasmid 1.

Au+

DETECTION Modeling DISPOSITION

The luxI system and luxI and luxR system turned out to have the same result.

IPTG

DETECTION Experiment DISPOSITION

The system involves both luxI and luxR has the better result.

IPTG

DISPOSITION DETECTION Modeling SUICIDE

The lysozyme will increase with time without IPTG and target molecules.

Au+

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+

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

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!

DETECTION Riboswitch

We used RNAfold to simulate the secondary structure of the ribozyme, aptamers and riboswitches.

MC

DETECTION Riboswitch MC7 C7 MC3 C31

Modeling results are used to screen out the best two riboswitches.

MC

DETECTION Sensor Specific promoter

for those we can find specific sensor

Riboswitch

for those without promoters

DEGRADATION DETECTION MlrD

The concentration of MC decreased faster with mlrD.

MC

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?

Polluted water Cleaner water Engineered bacteria

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.

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

Summary

Team

Practice&Policy

Team

Acknowledgment