The Smart EDC Farmland Protection System

In order to solve some real environmental challenges, our team has proposed an integrated system that can both detect and degrade endocrine disrupting chemicals (EDCs) suitable for farmland water protection.

When we started building this system, we aimed not only to solve a few farmers’ problems, but on a bigger scale, agricultural and industrial, since in most of the developing countries, factories could be easily found in between farmland. On the left-hand side of the slide, we developed the system that could sense the concentration of the EDC in the water and control the valve to protect the farmland from polluted water. And on the right-hand side of the slide, we could collect such data, such as the concentration, time, and place. If the number of devices could grow to dozens or say hundreds, we would be able to tell where and when did the pollution came from.

First, in normal days, the gate will remain half open and let the water comes into the channel. When the water passes through the gate, it will then passes through the filter. Enzyme mixed with activated carbon is filled in the filter, which can help filter out most of our target endocrine disrupting chemicals. We have proved the capability of the enzyme and filter through modeling and experiment test. Then the water will encounter out fluorescent detection. We pump some water into the detector and mix them with the indicator paper which is coated with modified E.coli, and the EDCs in the water can be captured by our modified E.coli. The E.coli is later excited with laser light to produce fluorescent, the fluorescent signal will be collected and calculated into relative EDCs concentration.

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If the detected EDCs concentration is safe for the farmland to use, the microcontroller which is embedded in the detector will send a signal to the gate to tell it to remain open to let the water in. However, if the detected EDCs concentration is above the safety standard, a feedback signal will be sent to the gate and lower it to protect the farmland from further damage. The reading of the EDCs concentration will also be sent to our database and the App, which can allow the farmer or the farmland manager to remotely monitor the condition of the farmland.

What is EDCs?

EDCs (Endocrine Disruptor Chemicals), which can interfere with endocrine systems, causing cancerous tumors, birth defects, and other developmental disorders. These chemicals are mostly man-made and found in various materials such as pesticides, food containers, and personal care products. In this project, we primarily focus on two common kinds of EDCs, BPA (Bisphenol A) and NP (Nonyl Phenol), in industrial wastewater.

We construct the gene of horseradish peroxidase (HRP) and transform 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 product.

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 very high 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 or surface plasmon resonance signal on the gold chip.

Source:Quantifying Hormone Disruptors with an Engineered Bacterial Biosensor. ACS Cent. Sci., 2017, 3 (2), pp 110–116

In the future, we will construct light detection system and optical system for surface plasmon resonance to complete our detection system. From observation of density of E.coli, it indicates that when the concentration of EDCs decreases, the density of E.coli decrease as well which coincide with our prediction.

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 human can bind estrogen and EDCs. The whole ER alpha is a huge protein, which means it may be difficult to expressed by E.coli. From previous research (Akiko Koide et al, 2001), the LBD can bind estrogen normally without other parts, so we chose its LBD to do 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 previous research (Akiko Koide et al, 2001), the type III domain from human fibronectin (FNfn10) can be used as monobody to probing whether ER alpha LBD bind to ligand.

BBa_K1694006:

This part is a gold binding peptide (GBP). We fuse our monobody and GBP so that the monobody can be displayed on the gold surface.

EDCs Degradation

There is three category of enzymes that is 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 of phenolic EDCs.

1.Laccase

BBa_K2354001 : Laccase Bp

This part encoded a laccase which originated from Bacillus pumilus. We codon optimize this part for E.coli and add a 6X histag at 3’ terminal to facilitate protein purification. In addition, we add a GS linker and a BamHI at 3’ terminal for possible protein fusion experiment in future.

BBa_K2354002 : Laccase Sc

This part encoded a laccase that previous study had codon optimized and construct on E.coli . We add a 6X histag at 3’ terminal to facilitate protein purification. In addition, we also add a BamHI at 3’ terminal for possible protein fusion experiment in future.

BBa_K2354003 : Laccase Tc

This part encoded a laccase which originated from Thanatephorus cucumeris. We codon optimize this part for E.coli and add a 6X histag at 3’ terminal to facilitate protein purification. In addition, we add a BamHI at 3’ terminal for possible protein fusion experiment in future.

BBa_K2354004: Laccase Tv

This part encoded a laccase which originated from Trametes versicolor. We codon optimize this part for E.coli and add a 6X histag at 3’ terminal to facilitate protein purification. In addition, we add a BamHI at 3’ terminal for possible protein fusion experiment in future.

2. HRP

BBa_K235400: HRP

This part encoded HRP ( horseradish peroxidase ) which originated from Armoracia rusticana. We codon optimize this part for E.coli and add a 6X histag at 3’ terminal to facilitate protein purification. In addition, we add a BamHI at 3’ terminal for possible protein fusion experiment in future.

3. MnP

BBa_K2354006 : MnP

This part encoded a MnP which originated from Trametes versicolor. We codon optimize this part for E.coli and add a 6X histag at 3’ terminal to facilitate protein purification. In addition, we add a GS linker and a BamHI at 3’ terminal for possible protein fusion experiment in future.