Description

We are aiming to degrade 3-phenoxybenzoate acid (3-PBA) thoroughly through employing Synthetic Biology concept and microbial biodegradation techniques. Also we are eager to take the first steps to make our project real.

Outline

1. Background - Why we need to degrade 3-PBA thoroughly?
2. Method - How to degrade 3-PBA thoroughly?
3. Practice - How to make our project real?

1.Background - Why we need to degrade 3-PBA thoroughly?

1. Harm to the enviroment and human
2. Drosophila toxicological experiment
3. Cost-benefit analysis for engineered bacteria

A.Harm to the enviroment and human

3-PBA is the main metabolite in the degradation of pyrethroids(widely used for insect control in agriculture industry). For being a metabolite, it is easy to be ignored. Through loads of information handling we list the main harm.

1. Long natural degradation half-life(180 days)
2. Hindering the mineralization of pyrethroids
3. Widespread occurrence in surface water, sediment, and soil
4. Potential reproductive toxicity

B.Drosophila toxicological experiment

C.Cost-benefit analysis for engineered bacteria

Degradation comes at a price.3-PBA thorough biodegradation is a series of enzymatic reactions. Protein synthesis and enzyme reactions needs materials、ATP and NADH. Catechol,the intermediate metabolite of 3-PBA, is toxic to the Escherichia coli. 3-pba as the carbon source can be degradated and absored entering the TCA circle to produce energy. Based on these facts we can get a formula Net proceeds(N)=Benefit(B)-Cost(C).

2.Method - How to degrade 3-PBA thoroughly?

1. Gene circut design
2. Experimental test & previous parts improvement & analysis
3. Gene circut redesign

A.Gene circut design

ptA: promoterA

ptB: promoterB

ptR: promoterRight

ptL: promoterLight

Before gene circut design, knowing the biodegradation process is necessary.

Thorough 3-PBA biodegradation means put 3-PBA into fumarate, pyruvate and HMS.

pbaA1A2B & pbaC gene cluster: coding degrading-enzyme to put 3-PBA into 3-hydroxybenzoate(3-HBA) and catechol

mhbDHIM gene cluster:coding degrading-enzyme to put 3-HBA into fumarate and pyruvate, carbon source

c230 gene fragment:coding degrading-enzyme to put catechol into HMS,ring opening product

mhbR gene fragment: coding operon mhbR

3-HBA is a kind of intermediate metabolite of 3-PBA but inducer. The promoterR expression strength is depend on the operon mhbR. Without the inducer 3-HBA the expression strength is of low level but when the inducer combines with mhbR, the expression strength is of high level. This makes that the degradation product is ued as an inducer for the expression of downsream genes.

To sum up, pba gene cluster codes degrading-enzyme to put 3-PBA into 3-HBA and catechol. 3-HBA works as an inducer making the high expression level of downsream genes real to put 3-HBA into fumarate and pyruvate used as carbon source. But at design stage the degradation of catechol was ignored.

B.Experimental test & previous parts improvement & analysis

To push the theory into the real, experimental test was demanded. During the experimental period some points put questions to us. After researching we decided to improve previous parts, CopA and RiboJ. For more details please read the Experiment section.

3.Practice - How to make our project real?

• Social investigation
• Applied design
• Concept & achievement sharing

The project came from life and finally will go back to life. For more details please read the Applied Design and Human Practice sections.