Team:NTHU Taiwan/Design
Design
The Smart EDC Farmland Protection System
In order to solve some real environmental challenges, our team has proposed an integrated system which is suitable for farmland water protection and can both detect and degrade endocrine disrupting chemicals (EDCs).
What is EDCs?
EDCs (Endocrine Disruptor Chemicals), which can interfere with our endocrine systems, causing cancerous tumors, birth defects, and other developmental disorders. These chemicals are mostly man-made and are found in various materials such as pesticides, food containers, and personal care products. Factories sometimes illegally emit the EDCs contaminated water into the river, which can post the threat to nearby farmland. In this project, we primarily focus on two common kinds of EDCs, BPA (Bisphenol A) and NP (Nonyl Phenol), in the agricultural irrigation systems.
How we degrade EDCs in the water?
We constructed the gene of horseradish peroxidase (HRP) and transformed it into E. coli. Horseradish peroxidase uses the iron-contained activate center to convert the phenolic group to free radicals and let them react with each other in the presence of hydrogen peroxide. Finally, we can get the less harmful products.
After producing horseradish peroxidase, we use a chemical method to combine the enzyme with activated carbon via covalent bonds. Since activated carbon has outstanding ability to capture organic compounds and it can help to accumulate EDCs in the higher concentration, the enzyme can degrade EDCs in a highly efficient way. Finally, we combine activated carbon/enzyme complex with our designed filter to complete all the degradation system.
How we detect EDCs in the water?
We will flow some freeze-dried E. coli and water samples through the gold chips. Since there are GFPs in the E. coli, if water samples which have passed our degradation system still containing EDCs. EDCs in the sample will combine with ER-alpha, causing the structure of ER-alpha changes. And then monobody will capture the bounded ER-alpha together with E. coli, leading to the change of fluorescence signal on the gold chips.
Biobrick Design
EDC detection
1.Express surface displayed ER-alpha LBD
BBa_J04500:
This is IPTG inducible promoter with RBS. We use this part to control gene expression.
(Designed by: Kristen DeCelle Group: iGEM2005)
BBa_K811005:
This is an INPNC part which can be used for the surface display of large proteins. We use this part to display our ER alpha LBD to bacteria surface.
(Designed by: Avin Veerakumar Group: iGEM12_Penn)
BBa_J18921:
This is GS-linker from iGEM. It can link two protein and make sure that each protein can fold normally.
(Designed by: Raik Gruenberg Group: Affiliates)
ER alpha LBD:
LBD means “Ligand Binding Domain”. This ER-alpha LBD from the human can bind estrogen and EDCs. The whole ER-alpha is a huge protein, which means it may be difficult to express by E. coli. From the previous research (Akiko Koide et al, 2001), the LBD can bind to estrogen normally without other parts, so we chose its LBD to conduct our experiment.
BBa_B0015:
A double terminator from iGEM. It can terminate DNA transcription.
(Designed by: Reshma Shetty Group: Antiquity)
2. Express Monobody with GBP
Monobody:
From the previous research (Akiko Koide et al, 2001), the type III domain from human fibronectin (FNfn10) can be used as monobody to probe whether ER-alpha LBD bind to the ligand(EDCs).
BBa_K1694006:
This part is a gold binding peptide (GBP). We fused our monobody and GBP so that the monobody can be displayed on the gold surface.
EDCs Degradation
There are three categories of enzymes that are reported to be capable of catalysis redox reaction with phenolic compounds. They are laccase, MnP (Manganese peroxidase), and HRP (horseradish peroxidase). We cloned several genes that encoded these enzymes into E. coli. We expressed the enzyme with the IPTG-inducible promoter (J04500), purified and tested the activity against phenolic EDCs to see if this enzyme can accelerate the degradation speed.
1.Laccase
BBa_K2354001 : Laccase Bp
This part encoded a laccase which originated from Bacillus pumilus. We codon optimized this part for E. coli and added a 6X histag at 3’ terminal to facilitate protein purification. In addition, we also added a GS linker and a BamHI at 3’ terminal for possible protein fusion experiment in the future.
BBa_K2354002 : Laccase Sc
This part encoded a laccase that the previous study had codon optimized and construct on E. coli. We added a 6X histag at 3’ terminal to facilitate protein purification. In addition, we also added a BamHI at 3’ terminal for possible protein fusion experiment in the future.
BBa_K2354003 : Laccase Tc
This part encoded a laccase which originated from Thanatephorus cucumeris. We codon optimized this part for E. coli and added a 6X histag at 3’ terminal to facilitate protein purification. In addition, we added a BamHI at 3’ terminal for possible protein fusion experiment in the future.
BBa_K2354004: Laccase Tv
This part encoded a laccase which originated from Trametes versicolor. We codon optimized this part for E. coli and added a 6X histag at 3’ terminal to facilitate protein purification. In addition, we added a BamHI at 3’ terminal for possible protein fusion experiment in the future.
2. HRP
BBa_K235400: HRP
This part encoded HRP ( horseradish peroxidase ) which originated from Armoracia rusticana. We codon optimized this part for E. coli and added a 6X histag at 3’ terminal to facilitate protein purification. In addition, we added a BamHI at 3’ terminal for possible protein fusion experiment in the future.
3. MnP
BBa_K2354006 : MnP
This part encoded a MnP which originated from Trametes versicolor. We codon optimized this part for E. coli and added a 6X histag at 3’ terminal to facilitate protein purification. In addition, we added a GS linker and a BamHI at 3’ terminal for possible protein fusion experiment in the future.
