Introduction
Our team has been focusing on food issues. Food is a basic necessity, however, COVID-19 and other human conflicts have brought devastating consequences. With the collapse in demand from restaurants, hotels, and catering, the closure of open markets, but a surge in demand from supermarkets during COVID-19, over 828 million people are suffering from hunger. It is painfully clear that we need a more sustainable and efficient farming system. Our team therefore set a target to improve current hydroponics and aim to establish a sustainable hydroponic agriculture system in the near future.
Hardware
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Research
Hydroponics system
Advantages
- Greater plant density- plants can be moved as they grow. The use of a growth room for germination and seedling production and the spacing of certain crops in the greenhouse decreases the average area needed per plant over conventional soil production.
- Higher yields- Reports of higher yields and better quality are common although equal yields should be able to be obtained from a conventional cropping system.
- Less water consumption- In methods where the root system is contained in a closed trough or tube, less evaporation occurs and water consumption is reduced.
Problem
However, the costs of hydroponics are high and it is unsustainable. In addition, since the capacity of the machine is large, an excessive amount of nutrition liquid is required to meet the concentration we want. Our experimental site is the school laboratory rather than a large laboratory, both space and resources will be less than in other laboratories which means that it is hard to produce such a large amount of the nutrition liquid.
Overall
Growth period: 55 to 60 days
Germination: 1 to 2 weeks
Temperature:
- Optimum temperature for germination: 20∼22°C
- Suitable temperature for growth: 20～30℃
- Sunlight time: 8 to 12 hours (the longer the dark period, the earlier the flowering)
- Optimum soil pH: 6.5 to 7.5
Brainstorm & Design
We would like to nvestigate possible methods to further reduce water usage/ waste in hydroponics.
Our team believes hydroponics to be a promising solution. However, since the capacity and the size of the hydroponics is large, an excessive amount of nutrition liquid is required. This will increase the costs of using hydroponics and it is less sustainable. Therefore, we came up with an idea to enhance urban hydroponics systems by redesigning the hydroponics.
Furthermore our experimental site is the school laboratory rather than a large laboratory, so both space and resources will be less than in other laboratories. This means that it is hard to place large hydroponics and produce such a large amount of the nutrition solution. This direction of improvement would allow us to finish our experiment more easily.
Build & Test (Phase I)
To fulfill our needs, we, therefore, try to use a smaller container, e.g. a beaker, to investigate if this, and the limited solution would eliminate the plant seeding. In our testing, the concentration of nutrient solution we aimed to have is 0.1 M. We used the hydroponic machine and beakers as apparatus and kept the same concentration. The table below illustrates that using the hydroponic machine requires 1 L of the nutrient solution and 10 L of water to mix up the targeted culture medium.Using the beaker as the vessel only requires 0.5 L of the nutrient solution and 5 L of water to get a 0.1M of concentration of the culture medium.
To stabilize the plant in the beaker, we used wooden sticks, as shown in Photo 2.
Learn & Improve (Phase I)
In our result, we showed that seeding was not eliminated. Using the hydroponic machine required 5L more water and 0.5L more nutrient solution than the beaker required. The culture medium used for the hydroponic cultivation of plants is required to be replaced regularly. Overall, using a smaller container as the planting vessel would be a more cost-effective way and less waste.
We understood that plant growth should be further investigated if the small solution volume would have an impact on this. However, since the target plant in the current project is not considered a large plant, we will focus on another issue: ‘Desipit using a breaker for our experiment is straightforward, having wooden sticks to stabilize the plant in the beaker is not user-friendly and hard to promote widely.
Build & Test (Phase II)
In recognised the current defect in our experiment kit, we designed a customized cover, and used a 3D printer to print this out. While designing this cover, we have to deal with several elements such as the thickness and the size. To fulfill the aim of the pH control in our project, we designed a pH detecting system and had our customized cover to accommodate this.
In this, our pH detecting system based on Arduino to connect with three pH meters and a Wi-Fi module from Dfrobot to monitor the pH value in real time. Also, we used three cups with a hole for the pH meter, a hole for plants, and a spot for the air pump. In order to make sure the other factors are the same, we connected all the air pump pipes together and put all the cups close together.
To investigate if our customized cover + pH detecting system is functional, we took the opportunity to place our plants and inoculate our engineered recombinant E.coli (transformed with glsA, pH shooting and pET11a as control) into the testing cup.
Factors
As the height of the plant container is fixed, it can be imagined that the thicker the cover, the higher the nutrition liquid level in the beaker to make the nutrition liquid seep into the sponge within the plant container.
In total, there are three holes in the cover for placing a plant container, pH sensor, and also water pump. The diameter of the holes is shown in the following figure. To prevent the vertically-placed pH sensor tilting or falling, we also design two additional supporters.
Learn & Improve (Phase II)
Through the current investigation, we have demonstrated that small container design is feasible in the hydroponic system, and with our E.coli pH control system, the water efficiency in our future hydroponics would be increased. However, in consideration of massive usage, the circular design of beakers has limited real world application.
After designing a gene to control the pH environment in the hydroponic system, we used data collected from the internet to build a model (model1), thus predicting how different pH values affect plant growth. The results showed that plants grow best in a neutral environment. Therefore, we decided to set up a pH monitoring system to monitor pH fluctuations every 10 minutes for a week.
Build & Test (Phase III)
We intend to optimize the current hardware. Through previous experiments, we found out that small containers are better than large ones, so we designed our hexagon-shaped kit.
This integrated kit was designed by hand drafting initially. We then made a three-dimensional model see if the design is workable or not. Afterward, we worked on various 2D components using Coreldraw and cut them out with a laser cutting machine using acrylic boards. Those components will be combined with acrylate adhesive and AB epoxy for the linking points to make it water-resistant.
Photo 8. The 2D design, including shapes, sizes and details of our home kit. (Right)
Photo 10. 3D rendering of the design of prototype home kit. (Right)
Learn and Improve (Phase III)
The kits can be mounted and arranged in a honeycomb pattern in order to fit in any shape. Home users can align numbers of them in their small balconies. In this way, we can encourage more people to engage in self-farming, promoting the idea of sustainable food production. In addition, by aligning thousands of them, we would have this portal device to be placed in abandoned buildings. The current design is easy to assemble and has an additional hole in the bottom to replace the solution if needed. Depending on the needs of the customers, the size of the current kit would be scaled up, and the upper half structure would be simpified.
We understood that plant growth should be further investigated if the small solution volume would have an impact on this. Due to the time limitation, we have yet to have any plant fully grow in this device then harvest it. Since the cycle II has demonstrated that the pH control would be nicely achieved by our engineered recombinant E.coli strains, we are interested to have follow up experiments to fully investigate the potential of our design and iGEM parts.
Protocol of the hydroponic kit
We use the lazer-cutter to cut the acrylic, and then we first mounted the bottom part using AB epoxy, and then we puzzle the upper part. After that, we use the adhensive to stick the LED belt on the upper cover, and weld the wires together. Eventually, we put the foam and the plant holder inside the middle part and pour water, nutrition liquid and transformed E.coli for testing.
Hormone Binding Proteins
- PYL8 (BBa_K4340601)
- NSP4-T2R4 (BBa_K4340600)
Hormone Binding Proteins is the second system we designed to accelerate the seed germination stage. Abscisic Acid (ABA) is a plant hormone that delays or stops seed germination in harsh environments (ie. extreme weather like droughts). In our indoor hydroponic setting, ABA is unnecessary and we want to stop its signaling to reduce the time required for seed germination. The system uses hormone-binding proteins PYL8 and T2R4. We have designed T2R4 with an NSP4 secretion peptide to increase the secretion of the protein out of the E.coli. PYL8, a new part, is a hypersensitive ABA binding protein that has a better ABA binding efficiency.
In our experiments, we submerged the soybeans in soft tissues absorbed with water to test out the effect of ABA and the function and efficiency of our plasmids in inhibiting ABA.
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Experiment 1: Hormone Soybean Tests
Since ABA is not soluble in water, we use DMSO (organic solvent) to dissolve ABA powder. Since DMSO is toxic to E.coli if the percentage is bigger than 1/100 in liquid [1] (Figure 1), in the first hour of the three groups of repeating experiments, 2 ul of ABA dissolved by DMSO in the ratio of 20mg/ml is added to the ABA treated groups (ABA, ABA-NSP4-ABA, ABA-PYL8, ABA-pET11a).
For all groups treated with our transformed E.coli culture, 60 ul of transformed E.coli culture is added at every checkpoint (09:00 in the morning during seven days.). All the surfaces of the soybean groups are covered with liquid by spraying distilled water at every checkpoint.
We tested whether the hormone binding domain can inhibit and attenuate the effect of ABA (plant hormone that inhibits germination and plant growth) to plant germination. The pET11a and PYL8 E.coli culture groups, and a group with ABA treatment and PYL8 E. coli culture had a higher germinated percentage than the group without ABA (blank). This indicated that the PYL8 had a positive effect on the seed germination stage both with ABA and without ABA. (Figure 2&3)
In the PYL8 group results, the percentage of germinated soybeans with PYL8 E.coli culture and ABA was significantly higher than in groups only with ABA. This shows that the PYL8 worked to produce an inhibitor of ABA. Notably, the PYL8 group (only added PYL8 cell culture) is even higher than the group without ABA. This demonstrated that PYL8 can discharge the effect of ABA, and even benefit seed germination squarely. (Figure 3)
NSP4-T2R4, however, had the same function as the pET11a vector control, (Figure 4) which means that NSP4-T2R4 might not have an outstanding effect on increasing the speed of germination of soybeans.
To sum up, the efficiency of the plasmids ranked is PYL8> NSP4-T2R4 (slightly higher than) pET11a.
Experiment 2: Western Blot and Real-time PCR
Figure 3: The real-time PCR result of NSP4-T2R4 comparing with BL21 E.coli strain. (Right)
We conducted a western blot experiment to validate the quality of protein expression of PYL8 and NSP4-T2R4. In the experiment, there is a clear band of PYL8 in both 10ul and 20ul samples at 35 kDa. There is a relatively more blurry band of NSP4-T2R4. (Figure 1)We conducted western blot experiment to validate the protein expression of PYL8 and NSP4-T2R4. In our experiment, there is a clear band of PYL8 in both 10ul and 20ul samples at 35 kDa. There is a relatively more blurry band of NSP4-T2R4.
In our real-time PCR test, the fold change of PYL8 against BL21 is lower than NSP4-T2R4. This means the mRNA of PYL8 is smaller than NSP4-T2R4 (while the DNA of NSP4-T2R4 is larger than PYL8), which is the same as predicted. (Figure 2&3)