Bio-SQUAD: BioSynthesis QUAntumDot
Yu-Cheng Chang¹, Chien-Chih Lin¹, Chien-En Huang¹, Yi-Ting Pan¹,
Ming-Hung Yen¹,
Manoj kandel¹, Yun-Fan Wang¹, Shun-Ping Huang¹,
Wun-Chien Huang¹, Syue-Hao Lyu¹,
Chen-Yu Weng¹, Jia-Ying Luo¹,
Yin-Erh Chen¹, Ya-Tang Yang²³§
¹iGEM Student Team Member, ²iGEM Team Mentor, ³iGEM Team Primary PI, §Faculty Sponsor, Department of Electrical Engineering, National Tsing Hua University, Hsinchu, Taiwan.
Team:NTHU Taiwan/Poster
Bio-SQUAD: BioSynthesis QUAntumDot
Abstract
We utilized the feature of biosythesis from the bacteria to solve the contamination of heavy metal in Hsinchu, where our university locate. In the mean time, we tried to produce the valuable products, quantum dots, in order to make the most of our project. Then, we used gene design and plasmid construction to elevate the efficiency of production and deal with the problem of survivability.
We built a bioreactor allowing the continuous monitor and measurement. Besides the experiments, we did the research and developed a model of our production of quantum dots.
We propagated our idea and project to different levels of education and hoped to awake their environmental awareness of protection and sustainability.
We built a bioreactor allowing the continuous monitor and measurement. Besides the experiments, we did the research and developed a model of our production of quantum dots.
We propagated our idea and project to different levels of education and hoped to awake their environmental awareness of protection and sustainability.
Problem
Quantum dots nowadays
The Defects of Chemical Synthesis
The multicolor property of QDs offer opportunities for a wide applications nowadays. For example, in vitro and in vivo bioimaging, targeting, and drug delivery have been widely discussed recent years. One of the most important QDs sensing application is heavy metal detection in environment. On top of these, QDs are important industrial material from TV displays to nanocomposites. Actually, quantum dots are now indispensable in technology and our daily life.
The Defects of Chemical Synthesis
However, the main way to synthesis quantum dots based on chemical methods, which includes: sol-gel Method, photochemical method, and solvothermal method. All the methods above require enormous solvents and need to be prepared in the high pressure and high-temperature environment and with only nitrogen. Solvent such as thioacetamide and polyvinylpyrrolidone are toxic or potential cancer causing agents. These factors are crucial for the exchange of ligands however result in a serious waste of energy and pollution. Especially photochemical preparation of quantum dots needing Co60 as resources that can not only cause radiation contamination but also end up endangering people’s health.
Idea
JM109
Part
cada
mnth
MT2
yhao
We chose E.Coli JM109 as our target organism due to its high efficiency in quantum dots synthesis indicated in many papers. Also the biosafety of JM109 is only level one to make sure a standard safe experiment environment is done.
Gene-BioSQUADThis year, our team NTHU_Taiwan would like to introduce innovative ways to increase the yield of quantum dots by our gene design. They are members of BioSQUAD, JM109, JM109 with yhaO, JM109 with MntH, JM109 with MT2 and JM109 with CadA. With this powerful squad, we obtain a much higher quantum dots amount.
Promoter CadA (BBa_K1724000) allows us to enhance the proteins production in the environment containing cadmium ions. Gene MntH(BBa_K1526007) allows the concentration of cadmium ions to be almost equal with extracellular and intercellular one. MT2 lowers the toxicity of cadmium ions, which is a heavy metal by using a peptide to combine with ions thus forming a chelate. Last but not least, yhaO(BBa_K2242233) can turn cysteine into hydrogen sulfide, which is a better ingredient for quantum dots synthesis.
Part
Here are all the parts we applied to compose a complete system for both BioSQUAD members also the hardware.
cada
Cada is a cadmium sensitive promoter, when cada combined with cadmium it will transcript the downstream gene.
To verify its function,we ligase RFP protein with it and detect the red fluorescent.The result shows that the cada-RFP has higher fluorescent than the control means that our promoter is work.
mnth
Actually cadmium doesn’t have its channel protein beacause it is toxin to organism.
But cadmium is smart that is can use other channel protein such zinc or Manganese channel.
And those heavy metal carry the same electron amount as cadmium so channel protein is hardly to distinguish their difference accurately.So we use mnth protein to help JM109 catch more cadmium to produce quantum dot.
MT2
Mnth will allow cadmium inside but too many cadmium will kill the JM109.Therefore we design MT to catch the cadmium.MT2 is kind of metallothionein protein that contain rich cys amino acid and it can use thiol group to Combine with 7 cadmium ions.
yhao
In order to introduce a synthetic biology and energy-saving way to manufacture quantum dots, we must increase the relative fluorescence value and the quantum rate. We use a gene named "yhao" as L-Cysteine desulfhydrases for enhancing the producibility.
Detection
To verify the production of quantum dot produced by bacteria we use multiple detection techniques. Firstly, we check the color change in bacterial solution by using UV transilluminator where change in color of different growth media was clearly observed.
Fig1. The fluorescence of medium under UV ray
After the successful verification of bacterial growth under metallic ion condition through visible color changer, next step was carried out under biotech instruments. Biotech microplate reader was used to measure OD value of bacteria culture. Once the desired OD value of 0.03 was reached, efficient bacterial growth under room temperature through gene editing under heavy metallic environment is proved.
OD value figure if we have
Next step is carried to authenticate quantum dot synthesis. Here, fluorescence detection technique is used whereby excitation of quantum dot inside bacteria using wavelength of 365 nm is used. This excites CdS ions to excitation state and shortly after which they release fluorescence wave of wavelength ranging from 400-600nm on coming back to ground state. The theoretical assumption match the experimental outcome proving quantum dot synthesis by genetically engineered bacteria.
Fig2. The mechanism of Quantum dots detection. (Fluorescence model)
Quantum dot is synthesized but where exactly is it taking place ? Is it extracellular? or intracellular? To answer this question we check fluorescence value of lysis solution and supernatant. The graph below clarifies "the fluorescence value of the lysis solution is much higher than that of the supernatant after centrifugation" suggesting intracellular synthesis of quantum dot.
Fig1. The fluorescence of medium under UV ray
After the successful verification of bacterial growth under metallic ion condition through visible color changer, next step was carried out under biotech instruments. Biotech microplate reader was used to measure OD value of bacteria culture. Once the desired OD value of 0.03 was reached, efficient bacterial growth under room temperature through gene editing under heavy metallic environment is proved.
OD value figure if we have
Next step is carried to authenticate quantum dot synthesis. Here, fluorescence detection technique is used whereby excitation of quantum dot inside bacteria using wavelength of 365 nm is used. This excites CdS ions to excitation state and shortly after which they release fluorescence wave of wavelength ranging from 400-600nm on coming back to ground state. The theoretical assumption match the experimental outcome proving quantum dot synthesis by genetically engineered bacteria.
Fig2. The mechanism of Quantum dots detection. (Fluorescence model)
Quantum dot is synthesized but where exactly is it taking place ? Is it extracellular? or intracellular? To answer this question we check fluorescence value of lysis solution and supernatant. The graph below clarifies "the fluorescence value of the lysis solution is much higher than that of the supernatant after centrifugation" suggesting intracellular synthesis of quantum dot.
Hardware
To address the lack of accessibility to implement OD and fluorescence measurements due to the high costs and the level of knowledge required among others, our team has designed a bioreactor, a fully-equipped bioengineering device capable of automatically monitoring biosynthesizing quantum dots' process.
To ensure a functional, economical and easy-to-use design, collaboration between the hardware and the wet lab teams has been necessary throughout the development process. After several discussion with Professor YA-TANG YANG, we adopted a unique design that which can be easily transported and replicated at a cost not exceeding $60.
Figure1. The overview of BIOSQUAD bioreactor.
Figure 2. OD calibration curve.
To monitor bacteria's growth, we need to measure optical density (OD) value and fluorescence, thus we implement a high precision light sensor we decided to use BIR-BM13E4G-2 and SUB/S530. This converts the magnitude of light received into voltage, which can be precisely measured. Since the voltage is not the real OD value, therefore, we did several experiments to calibrate it. With the calibration curve, we now can tell the difference in the culturing process.
To ensure a functional, economical and easy-to-use design, collaboration between the hardware and the wet lab teams has been necessary throughout the development process. After several discussion with Professor YA-TANG YANG, we adopted a unique design that which can be easily transported and replicated at a cost not exceeding $60.
Figure1. The overview of BIOSQUAD bioreactor.
Figure 2. OD calibration curve.
Modeling
On observing the fluorescence spectrum, we can find out that there are two peaks as shown in figure below.
Fig.1: The fluorescence spectrum of quantum dots is showed above. There are two peaks in spectrum.
We have a deduction which is that the quantum dots we synthesis have at least two different size. However, we want the size of quantum dots to be about the same. What cause this phenomenon is the two pathway for JM109 to s ynthesis quantum dots, extracellular and intracellular.
We will do a simulation of homogeneous nucleation to find out what cause this phenomenon. The model we use here is phase field model. We change the concentration of Cd solution (solution mean density) and find the inflection point of free energy versus time which influence the nucleation speed.
We plot the time needed to reach the inflection point versus mean density and do a fitting.
fitting curve:
We find out that the inflection point and the mean density have the exponential growth relationship, which means that the solution concentration influence nucleation speed obviously. Since JM109 does not have specialized Cd channel, the simplest way to make the size of quantum dots uniformity is to introduce a Cd channel protein, mnth, to balance the extracellular Cd concentration and intracellular Cd concentration.
Fig.1: The fluorescence spectrum of quantum dots is showed above. There are two peaks in spectrum.
We have a deduction which is that the quantum dots we synthesis have at least two different size. However, we want the size of quantum dots to be about the same. What cause this phenomenon is the two pathway for JM109 to s ynthesis quantum dots, extracellular and intracellular.
We will do a simulation of homogeneous nucleation to find out what cause this phenomenon. The model we use here is phase field model. We change the concentration of Cd solution (solution mean density) and find the inflection point of free energy versus time which influence the nucleation speed.
We plot the time needed to reach the inflection point versus mean density and do a fitting.
fitting curve:
We find out that the inflection point and the mean density have the exponential growth relationship, which means that the solution concentration influence nucleation speed obviously. Since JM109 does not have specialized Cd channel, the simplest way to make the size of quantum dots uniformity is to introduce a Cd channel protein, mnth, to balance the extracellular Cd concentration and intracellular Cd concentration.
Conclusion
Successfully transfer the target gene(cada-yhaO/mnth/MT-2) into the target strain(E.coli JM109)
idea:
detection:
modeling:
- Basically confirm the heavy metal toxicity resistance function of MT-2 peptide.
- Successfully improve the QD production capacity of the strain (cada-yhaO).
- Basically confirm the feasibility of fluorescence detection method (UV light) (for QD generation).
- Basically confirm that the main place for QD generation is in the cell.
idea:
- Successfully cloning of four genes.
- Verify each gene's function.
- Successfully enhancing the producibility of quantum dot.
detection:
- We can use fluorescence to check QD production. (activated by UV ray).
- Most of QDs are synthesized in the cells (intracellular synthesis).
modeling:
- To make the size of quantum dots uniformity, We introduce a Cd channel protein, mnth.
Future Work
- According to our market research, 88.6% Taiwanese and 61.5% global participants indicate that they will prefer environmentally friendly biosynthesized quantum dots over normal ones.
- At the same time, participants gave an average point of 3.5 out of a scale of 5 on how much they think they are aware of heavy metal water pollution issue. Participants also leave comments as the following: "It is a problem well known but bearly address in Peru since there are conflict of interests of the companies that pollute the environment.”
- As the Taiwanese environmental activist, Jui-Kuang Chao, mentioned that the lack in public awareness is one of the reasons heavy metal water pollution issue is not yet tackled, we hope to combine our BioSQUAD project with that.
- We interviewed Prof. Hsueh-Shih Chen, an expert in quantum dots manufacturing. He told us about the possibility of creating a heavy metal pollution sensor out from our biosynthesized quantum dots.
- As a result, we aim to develop a product that could help sense heavy metal pollution in water, which we think is a feasible solution for raising the public’s awareness on heavy metal water pollution issue.
- We then did another survey on asking the public whether our idea can help them be more aware of the heavy metal water pollution around them. Surprisingly, 100% of the participants indicate that they think our product can help!
“I think mass media doesn't inform that much about heavy metal pollution to the public.”