Team:OSA/Poster

Poster: OSA

METzyme: A DNAzyme-based Heavy Metal Ion Detection Kit

Team Members:

Angel Hu, Jacqueline Yang, Oscar Ji, Henry Pan, Jerry Huang, Oscar Sun, Michael Liu, Ke Xu, Teddy Meng, Terry Wo, Molinia Yang, Vivian Zhang, Eric Zhang, Eric Ding, Louie Lv, Tiger Ding

Abstract:

High-density metals like lead and mercury, also known as heavy metals, have severely harmed the environment and our bodies. Every year heavy metal exposure results in millions of disabilities worldwide. Surprisedly, previous detecting methods for this urgent issue either require bulky instruments or professional technicians. Thus, it is imminent for us to devise a cheap, portable, accessible, accurate detection kit. We use DNAzyme, which can cleave ssDNA when combined with Zn2+, Mn2+, Pb2+. Cu2+. To maximize outputs, four amplification systems (HTDC, Toehold Switch, TO-DNA, and CRISPR-Cas12a) are also used. While we test these theories in lab, our human practice team interviews experts and discovers such a kit is not only needed for water detection but also for soil, which becomes the new focus of our project. We have achieved fluorescence and lateral flow detection of heavy metal ions which contribute to alleviating this severe environmental issue worldwide.

Inspiration

Heavy metal ions are extraordinarily harmful to the human body and to the environment. We take inspiration from the contamination of heavy metal ions in both aquatic and terrestrial ecosystems. Heavy metal ions will enter soil and water bodies, then cause the loss of soil fertility. It will also enter the food chain through animals and plants and finally into our bodies. We did some relevant industry interviews and experts help us to relocate and specify our project’s importance.

Fig.1 The three step of how heavy metal ions enter the ecosystem

Project Goal

After discovering this is a severe issue, we did some research about the previous methods that scientists have developed to solve it. The truth is these methods either require bulky instruments or professional technicians. Considering the urgency of this issue, we believe there is need to develop an accurate and efficient detection system.

Fig.2 The two disadvantages of previous detecting systems

DNAzyme

Through these investigations, we clearly see the need of a fast and efficient heavy metal detecting method. By reading various articles, we discovered a property of DNAzyme that makes it suitable as a detecting technique: it can cleave towards its substrate after binding to its target heavy metal ion. So we conducted experiments on three DNAzymes: 8-17, F8-X, and S9 DNAzyme.

Fig.3 A. DNAzyme mechanism B. DNAzyme sequence

We conducted experiments of 8-17 DNAzyme under different Pb2+ concentrations, F8-X DNAzyme under different Cu2+ concentrations, S9 DNAzyme under different Mn2+ concentrations.

Fig.4 DNAzyme testing.

We used the microplate reader to detect the fluorescence signal.

A: 8-17 DNAzyme detection limit testing
B: F8-X Cu2+ detection limit testing
C: S9 Mn2+ detection limit testing
D: DNAzyme testing summary

To achieve higher efficiency, we conducted DNAzyme mutation test and successfully discovered a mutant that has higher activity.

Fig.5 DNAzyme mutation testing.

Signal Amplification

In order to further improve the working efficiency of DNAzyme, we incorporated the design of Signal Amplification. Through research, we found four systems to amplify the input signals: HTDC, TO-DNA, Cas12a, and Toehold Switch. Out of the four, HTDC and Cas12a proved to be the most efficient in detecting heavy metal ions.

Fig.6 HTDC Detection Limit

We used a Microplate Reader to detect the fluorescence signals.

A: Pb2+ 1μm
B: Zn2+ 300μm
C: Mn2+ 100 μm

The figure below indicates the CRISPR Cas12a Combination Testing with S9 DNAzyme showing fluorescence values. When the heavy metal ion is attached to the S9 DNAzyme, the substrate strand will be cut at the cleavage site. The rising trend shows success in detection.

Fig.7 CRISPR Cas12a Combination Testing

Model

The purpose of our modeling is to understand the status of the CRISPR-Cas12a system in our project and determine the effect of different lengths of ssDNA. To achieve this goal, we utilized the Michaelis-Menten Equation to analyze the reaction.

Fig.8 Michaelis–Menten Equation of Cas12a reaction.

A: Michaelis–Menten Equation of Cas12a.
B: Reaction rate V relating to concentration of substrate S.
C: Lineweaver-Burk Equation.
D: An example of Lineweaver-Burk Plot.

Then we made another equation. “a” represents fluorescence intensity, “t” represents time, “θ”refers to “V”, and when t=0, θ refers to V0. Based on this equation, we can determine the factors in the Michaelis-Menten equation.



Through our experiment data, we acquired the values of “a” and “t”, thus determining “V0”. We then use the value of “V0” as well as “S” to find the value of “Km” and “Vmax”.

Fig.9
Vmax & Km Value of CRISPR-Cas12a experiment based on Michaelis–Menten equation.

A: Michaelis–Menten saturation curve for Cas12a Enzyme with 11 nt, 13nt, 15nt;
B: Vmax and Km value of 11 nt, 13 nt, 15 nt ssDNA CRISPR-Cas12a experiment.

The “Km” and “Vmax” value of 11nt are higher. This indicates that a short ssDNA can prompt the reaction faster. However, longer chain has better gRNA binding capacity, that is, it is more difficult to dissociate after being bound to the Cas12a complex. So we eventually selected ssDNA-15nt.

iHP

With the aim of identifying the industrial value of out product, we visited 3 companies. Through these interviews, we recognized that there is a customer demand for the population, but not necessarily for industries. After attending multiple field trips and interviews, we identified that for industries, the demand is more toward heavy metal regulation in a soil base instead of water.

Fig.10 Companies we intereived

Fig.11 Interview on Lejieshi environmental technology

Moreover, through interviewing senior experts in the field, we learned that it is very important to have humanistic care in which we need be aware that the public would value the simplicity and convenience of our product.

Fig.12 Professor Long Co-chairman of Oriental Low-carbon Corporation, Professor of Huadong Shifan University

Project Modification

After knowing the market condition and our stake-holders needs, we started to improve our project according to these suggestions. To provide customers with a convenient and low-cost detecting method, we transformed HTDC system to the form of test strip. Instead of operating bulky and expensive apparatus, users can test the target liquid whenever and wherever they want.

Fig.13 HTDC Combined with Lateral Flow Assay.

Left: Pb2+ Positive
Right: Pb2+ Negative

Moreover, we decided to have an additional experiment on detecting the heavy metal ions in soil. However, despite several attempts and hard workings, we are still unable to apply our detection system in a soil base due to the fact that we're enable to extract heavy-metal ions from a soil base.

Fig.14 HTDC soil-base detection.

Left: Simulated Soil Sample
Right: Heavy Metal Ion Extract

Public Engagements

For our public engagement, we created an official Account of OSA on WeChat and posted our progress on a weekly basis.

Fig.15 Public account video shooting.

Our team also set up a booth in a social fair in Shanghai, China. We aim to raise awareness for heavy metal pollution and advocate for our iGem project.

Fig.17 Figure of Exhibition.

Parts

We conducted an in-depth mutation test of the 8-17 DNAzyme. We manually designed 95 sequences and discovered several of them, such as Mut 10, to be considerably more efficient than the original sequence. We also repeated the test three times.

Fig.17 8-17 DNAzyme mutation test.

Fig.18 Further testing of 14 candidate mutations.

In addition to the mutation experiment, we also uploaded a lot of parts, which are involved in multiple experiments, such as: DNAzyme, CRISPR-Cas12a, HTDC, etc.

Fig.19 The parts we submitted besides our Parts Collection

Results & Contribution

We are not the first team to focus on DNAzyme. Before us, several iGEM teams have used DNAzyme in their projects. Among these teams, the concept of "Hot Metal Switch" by the Pittsburgh team in 2016 is very similar to our project. They used DNAzyme 8-17 as a heavy metal ion detector. But our project is also different from theirs: we have conducted a specific and in-depth study of the 8-17DNAzyme used, and contributed a lot of new parts to iGEM. We will once again introduce the project.

- Lab: an in-depth study of the 8-17DNAzyme; contributed new parts to igem;

- We realized the detection of heavy metal ions by its ability to bind heavy metal ions and cut DNA single strands.

- We have done a mutation test of the 8-17DNAzyme and selected out mut-10 through several rounds of elimination; We have also created a corresponding model calculating the relative fluorescence of each mutant.

- HP: raise public awareness, collaboration with industries, better the human condition;

- Model: built a model around Cas 12a reaction

- We also established applications to the real world.

Acknowledgement & Sponsor

Thank you to Tolobio for providing us with Cas-12a nuclease, thank you to One Step Ahead and Bluepha Lab for accommodating and supporting us, thank you to SnapGene for creating their awesome app and providing us with a free license, thank you to The Hockaday School, Wilbraham & Monson Academy, Shanghai Starriver Bilingual School, YK Pao School, Shanghai High School International Division, Southwest Weiyu Middle School, Cardigan Mountain School, St. Andrew’s School(DE.), The Hun School of Princeton, Georgetown Prep School, St. Paul’s School, The Webb School, Suffield Academy for supporting us in this beautiful, invaluable iGEM community.

Fig.20 Acknowledgements & Sponsor