iGEM 2021 — Team Yonsei_Korea

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# Team:Yonsei Korea/Notebook

IGEM\_YONSEI

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In the Laboratory:

The **Science**
Behind It All

NOTEBOOK

DAILY RECORD

The full record in pdf format can be found [here.](https://static.igem.org/mediawiki/2021/4/4c/T--Yonsei_Korea--note_book.pdf)

YONSEI\_KOREA 2021
![](https://static.igem.org/mediawiki/2021/b/b7/T--Yonsei_Korea--main_logo_1.png)

Yonsei\_Korea is a convergent team of students based in Seoul, South Korea striving to create meaningful change utilizing synthtic biology in the iGEM 2021 Competition.

* NoteBook

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# Team:Yonsei Korea/Engineering

IGEM\_YONSEI

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In the Laboratory:

The **Science**
Behind It All

ENGINEERING SUCCESS

OVERVIEW

    To develop a diagnosis tool in detecting the *Magnaporthe oryzae* fungi, we incorporated engineering along with synthetic biology into our research. Our goal was to develop a proof of concept for a detection system, which is simple but specific.
Also, we aimed to design a tool that is easy to use and visualize, so that many people will be able to use it. Therefore, it was essential for us to incorporate *the engineering design cycle: research → imagine (brainstorming) → design → test → learn & improve*. We have optimized this in a way that is suitable and practical for our research.

    We divided our research into *two phases*, and went through *several iterations of the engineering design cycle for each phase*. *Phase 1* aimed to apply synthetic biology to develop a system that could specifically detect the Magnaporthe oryzae gene. *Phase 2* aimed to apply nanoscience and technology to develop a colorimetry tool to visualize the specific detection, which is important for practical purposes.

Through our stages of the engineering design cycle, we have achieved the following:

1. 1. DNAzyme specifically detects the mif23 gene of Magnaporthe oryzae.
2. 2. The detection system based on DNAzymes is effective, as DNAzyme cleavage activity is proportional to the amount of the substrate gene.
3. 3. DNAzyme is specifically catalyzed by Cu2+ ions.
4. 4. Gold nanoparticles are functionalized with the complementary sequences to our target gene.

PHASE ONE

Phase 1: Applying synthetic biology in developing a specific detection system for Magnaporthe oryzae

1st cycle

*1. Research*
    After acknowledging that Magnaporthe oryzae and rice blast disease are severe issues in rice plantations, we started research on the current diagnosis and treatment methods of the rice blast disease. We found that current detection systems rely on Artificial Intelligence (AI) technologies (Chen, Ling, et al.) or PCR-based methods. Although these methods demonstrate rapid detection, we have found out that they are skill extensive or show late diagnosis. In addition, they are expensive, so farmers are not able to utilize current technologies. Current treatment methods have many potential problems as well, as farmers rely heavily on chemical fungicides (Magar, Acharya, et al.). Chemical fungicides are sprayed extensively at each stage of rice growth, causing pollution and posing a risk in the ecosystem, as they are highly toxic.

    To develop a nucleic acid-based diagnosis tool, we researched more on PCR-based detection systems, to get an idea about which genes can be used as a target for our detection system. The following is what we found as potential targets for detection:

| Gene | PCR method | Reference |
| --- | --- | --- |
| Alb 1 superfamily hypothetical protein MGG\_04322 | LAMP, q-LAMP | Li, L., Zhang, S. Y., & Zhang, C. Q. (2019) |
| mif23 gene | Conventional PCR | Chadha, S., & Gopalakrishna, T. (2006) |
| 18S-28S region of rDNA | SYBR Green I qPCR | Sun, G., Liu, J., et al. (2015) |
| AVR genes | Conventional PCR | Selisana, S. M., Yanoria, M. J., et al. (2017) |

*2. Imagine (brainstorming)*
    Based on the research, we started brainstorming the factors that should be considered in designing our diagnosis tool. First, it should be simple and easy to use. We thought that current technologies such as PCR are not practical for farmers to use, so we tried to incorporate DNAzymes into our research. Second, specific detection should take place. To do this, our system should target genes that are specific to M. oryzae. Third, it should be visualized with the naked eye: it would not be practical to use expensive and complicated software or machines, nor need specific environmental conditions. We drafted ideas regarding colorimetry analysis, such as incorporating fluorescence or nanoparticles to show color change. Lastly, it should be affordable. To accomplish this, we thought of ideas that use the least amount of materials and equipment possible. This would not only reduce the price, but would also enhance the simplicity of the detection system.

Through this stage of brainstorming, we established the goal for the design phase: using DNAzymes that can specifically detect a unique gene of M. oryzae, and observing this activity with gold nanoparticles (colorimetry analysis). We incorporated DNAzymes into our idea, mainly because it would enhance the simplicity of our detection system, but not decrease specificity at the same time. Unlike many PCR-based detection methods, DNAzymes do not require DNA amplification and do not use specific enzymes. Therefore, complex buffer conditions and skills to use advanced instrumentations are not required. At the same time, it has high specificity, since it only exercises cleavage activity when the left and right binding arms of DNAzyme are properly bound to the target DNA. As we are aiming to develop a proof of concept that is simple and easy to use for farmers, we thought that DNAzymes would be a great idea in this context.

*3. Design*
    We incorporated the F-8 DNAzyme into our research to detect the unique gene of M. oryzae. Based on our research, we selected the mif23 gene as the target.

As the F-8 DNAzyme specifically catalyzes the cleavage at the thymidine of the YTGC (Y=T/C) sequence, we found potential regions of the mif23 gene (Genbank ID: GI 4732021) where the F-8 DNAzyme could bind and cleave it. Based on this, we designed 4 possible DNAzymes.

*[mif23 gene]*

| Name | Sequence (5’→3’) |
| --- | --- |
| Gene A | ACGGCCAGTGCCGGCGACAGCTCTAGCAACCCCACTGGCTCGGCTGCCTCCGTCACCAAATCCGGGTCAGGACCGCGGGAGACAAACT |
| Gene B | CAATCGACGAGACAATGCATTCCAACACAGTCCTGTTCATGATTGCCGCGGCGACGGGCGTCATGTCGGCCGAACTCAACCTCTTCCC |
| Gene C | CTCCTAACCGTGCCTACCAGACCAACGCATGCTCAATCACTGTTGCCGCCTCGGTATCGGATAAGTTCAACGCATACACAAGCACTAT |
| Gene D | CGGGCCATGCCGTCGTCAGACGTGAGGGTGCAGCTCGCGAGACTGCGAGCCCCTCGCTACCAAGTATCAATGACCCTTGTCTGGCGAG |
| DNAzyme A | AGTTTGTCTCCCGCGGTCCTGACCCGGATTTGGTGACGGAGGTGGATGCCGGGTCCGCCGAGCCAGTGGGGTTGCTAGAGCTGTCGCCGGCACTGGCCGT |
| DNAzyme B | GGGAAGAGGTTGAGTTCGGCCGACATGACGCCCGTCGCCGCGGTGGATGCCGGGTCCATCATGAACAGGACTGTGTTGGAATGCATTGTCTCGTCGATTG |
| DNAzyme C | ATAGTGCTTGTGTATGCGTTGAACTTATCCGATACCGAGGCGGTGGATGCCGGGTCCACAGTGATTGAGCATGCGTTGGTCTGGTAGGCACGGTTAGGAG |
| DNAzyme D | CTCGCCAGACAAGGGTCATTAGTACTTGGTAGCGAGGGGCTCGTGGATGCCGGGTCCGTCTCGCGAGCTGCACCCTCACGTCTGACGACGGCATGGCCCG |

    For example, our DNAzyme A would cleave the target Gene A based on the following mechanism:

Gene A
5’-acggccagtgccggcgacagctctagcaaccccactggctcggctgcctccgtcaccaaatccgggtcaggaccgcgggagacaaact-3’

DNAzyme A
3’-tgccggtcacggccgctgtcgagatcgttggggtgaccgagccgcctgggccgtaggtggaggcagtggtttaggcccagtcctggcgccctctgtttga-5’

1. ●
   The blue highlighted ctgc sequence in Gene A is specifically recognized by the DNAzymeA. Among the “CTGC”, T (thymidine) is where DNAzymeA specifically exercises its catalytic activity.
2. ●
   The purple highlighted gcctgggccgtaggtg sequence represents the loop structure of the DNAzyme. On the left is the left-binding arm (green), and on the right is the right-binding arm (pink). As described in the figure below, hybridization between complementary base pairs occurs. The left-binding arm can hybridize with the green sequence in Gene A, and the right-binding arm can hybridize with the pink sequence in Gene A.
3. ●
   Therefore, DNAzymeA and target GeneA would interact as:

![igem_yonsei](https://static.igem.org/mediawiki/2021/1/1e/T--Yonsei_Korea--success_1.png)

    In addition, in order for the DNAzymeA to be activated, important ions such as Cu2+ is essential. Therefore, with the addition of Cu2+, the target Gene A will be cleaved as below:

Gene A
5’-acggccagtgccggcgacagctctagcaaccccactggctcgg
ctgc
ctccgtcaccaaatccgggtcaggaccgcgggagacaaact-3’

DNAzyme A
3’-tgccggtcacggccgctgtcgagatcgttggggtgaccgagcc
gcctgggccgtaggtggaggcagtggtttaggcccagtcctggcgccctctgtttga-5’

![igem_yonsei](https://static.igem.org/mediawiki/2021/8/8e/T--Yonsei_Korea--success_2.png)

In designing our experiment, we added 3 controls. Therefore, 4 different types of conditions were tested in one experiment.

| 1 | DNAzyme only |
| --- | --- |
| 2 | Gene (substrate) only |
| 3 | DNAzyme + gene, but without catalyzing the cleavage reaction |
| 4 | DNAzyme + gene, with catalyzing the cleavage reaction |

*4. Test*
The synthetic DNA was ordered from BIONICS, a biotechnology company based in Korea.

    We designed the cleavage assay experiments based on the protocol provided by Wang, Zhang, et al. The difference between 3 and 4 is whether the reaction buffer was added. Therefore, in 3, the DNAzyme-substrate is expected to hybridize with each other, but it will not be cleaved. In contrast in 4, cleavage activity is expected to be shown as the reaction buffer is added.

| Sample | 1 | 2 | 3 | 4 |
| --- | --- | --- | --- | --- |
| 10 µM DNAzyme | 2 µL | - | 2 µL | 2 µL |
| 1 µM Substrate | - | 2 µL | 2 µL | 2 µL |
| Ions (2x reaction buffer) | 2 µL | 2 µL | - | 2 µL |
| 100mM HEPES (pH 7.4) | - | - | 2 µL | - |
| Milli-Q water | 16 µL | 16 µL | 14 µL | 14 µL |

    To ensure proper and specific hybridization between the DNAzyme and the substrate gene, the DNAzyme-substrate mixture was heated at 90°C for 5 minutes and cooled at 25°C (room temperature) for 10-15 minutes. The cleavage assay was then tested for DNAzyme-substrate A and B. Thereafter, the samples were incubated at 37°C for 16 hours for the cleavage reaction to occur. Finally, EDTA was added to stop the cleavage reaction.

    To visualize the results, agarose gel electrophoresis was performed. We optimized the agarose gel electrophoresis by trying at different agarose concentrations (1%, 1.2%, 2%), different gel running times (30 minutes, 10 minutes, every 10 minutes for 30 minutes), and different voltages (100V, 135V, 200V). To ensure that the DNA were properly stained by SYBR Green, we viewed it under the UV light as well. In addition, Nanodrop was done to ensure the DNA concentration and purity.

*5. Learn & Improve*
    From the agarose gel electrophoresis results, we were able to check that the hybridization between the DNAzyme and gene substrate occurs, by comparing samples 1 and 2 with sample 3: compared to the bands for samples 1 and 2 (which only contain either DNAzyme or substrate), the bands for sample 3 was thicker, which suggests that hybridization occurred. It seemed that the cleavage activity occurred when looking at sample 4, because the intensity of the band is weaker, compared to sample 3.

![igem_yonsei](https://static.igem.org/mediawiki/2021/e/ed/T--Yonsei_Korea--success_3.png)

    However, the main problem was that the band for sample 2 were not appearing for all cases: both gene A and gene B were not appearing as a single band. DNA was properly stained when it was viewed under the UV light, so we assumed that other experiment methods other than agarose gel electrophoresis should be used to view the results. Also, although the intensity of the band at 4 decreased, we were not seeing expected bands which represent the cleaved products. We were expecting to see bands around 40 bp, but we were not able to visualize them.

Another concern was that the DNA purity was not good, as the Nanodrop results show. For instance, the 260/280 ratio of sample 1 - 4 from DNAzyme-substrate B were all above 1.8, which indicates that DNA in all of the samples are not pure (low purity). This suggests that the quality of the DNA might be low, which might interfere with the DNAzyme cleavage activity, as F-8 DNAzyme cleavage is highly dependent on the exact sequence: at the thymidine of YTGC (Y=T/C) sequence.

    Table 1: Nanodrop analysis with DNAzyme-substrate B (August 26th, 2021)

| Sample | B-1 | B-2 | B-3 | B-4 |
| --- | --- | --- | --- | --- |
| 260/280 ratio | 2.405 | 4.072 | 2.283 | 2.257 |

    For improving our research for the 2nd cycle, we need to consider the following:

1. (1) Another experiment method for better visualization of bands
2. (2) Add controls to optimize the experiment process and see how each procedure in the experiment affects the cleavage reaction

2nd cycle

*1. Research*
    In the 2nd cycle, we aimed to improve the visualization of the bands. Therefore, we researched other experiment methods other than agarose gel electrophoresis. We have found that native PAGE and denaturing PAGE were also used frequently. Agarose gels are used for large fragments of DNA, whereas polyacrylamide gels are better to use for shorter lengths of DNA. The difference between native and denaturing PAGE was that denaturing PAGE uses urea, so that double-stranded DNA will be separated into single-stranded DNA.

*2. Imagine (brainstorming)*
    Since our target gene is 88bp and DNAzyme is 100bp in length, we thought that PAGE would be a more suitable method for our experiment. Also, the cleaved gene products are about 40bp in length, so agarose gel electrophoresis would not be the best in visualizing. In addition to brainstorming about experiment methods, we have found the need to add more controls to optimize the experiment process and see how each procedure in the experiment affects the cleavage reaction.

*3. Design*
    In the 2nd cycle, we added two additional controls: how EDTA and heat incubation at 90°C affects the cleavage reaction.

1. ● Sample 5: Equal to 4, but without EDTA. Since EDTA is not added, the ions present in the reaction buffer will not be chelated. In other words, since the ions are still present after 16 hours of incubation at 37°C, the DNAzyme will still be active. As a result, DNAzyme will be able to cleave the target substrate.
2. ● Sample 6: Equal to 4, but without the heat incubation at 90°C.

| 1 | DNAzyme only |
| --- | --- |
| 2 | Gene (substrate) only |
| 3 | DNAzyme + gene, but without catalyzing the cleavage reaction |
| 4 | DNAzyme + gene, with catalyzing the cleavage reaction |
| 5 | DNAzyme + gene, catalyzing the cleavage reaction, but without stopping it with EDTA |
| 6 | DNAzyme + gene, catalyzing the cleavage reaction, but without heat incubation at 90°C |

*4. Test*
The synthetic DNA was ordered from BIONICS.

    We designed the cleavage assay experiments based on the protocol provided by Wang, Zhang, et al. The experiment procedure is the same as in the 1st cycle.

| Ladder | B-1 | B-2 | B-3 | B-4 | B-5 | B-6 |
| --- | --- | --- | --- | --- | --- | --- |
| 10 µM DNAzyme | 2 µL | - | 2 µL | 2 µL | 2 µL | 2 µL |
| 1 µM Substrate | - | 2 µL | 2 µL | 2 µL | 2 µL | 2 µL |
| Ions (2x reaction buffer) | 2 µL | 2 µL | - | 2 µL | 2 µL | 2 µL |
| 100mM HEPES (pH 7.4) | - | - | 2 µL | - | - | - |
| Milli-Q water | 16 µL | 16 µL | 14 µL | 14 µL | 14 µL | 14 µL |
| Heat incubation | O | O | O | O | O | X |
| EDTA | 1 µL | 1 µL | 1 µL | 1 µL | - | 1 µL |

    To visualize the results, native PAGE was performed. 12% PAGE was prepared, and the gel was run at 900V for 60 minutes.

*5. Learn & Improve*

![igem_yonsei](https://static.igem.org/mediawiki/2021/d/d4/T--Yonsei_Korea--success_4.png)

    While using PAGE, sample 2 was visible, which was not possible to observe while performing agarose gel electrophoresis. In addition, we were able to check that the hybridization between the DNAzyme and gene substrate occurs, by comparing sample 1 and 2 with sample 3. In sample 3, there are two bands: the upper band represents the hybridized DNAzyme-substrate complex and the lower band represents the DNAzyme that is not hybridizing with the gene. This should happen because we inserted 10 times more concentration of DNAzyme compared to the concentration of the gene substrate. However, it was still hard to clearly see the cleavage activity. Comparing samples 3 and 4, it is hard to see the difference in the bands, although it seems like the intensity of the lower band is slightly weaker in sample 4, compared to sample 3. In addition, the PAGE results of sample 4 ~ 6 are similar: it is hard to visualize the difference between them.

    For improving our research for the 3rd cycle, we need to consider the following:

1. (1) Test with the sequence that is present in the reference paper (Wang, Zhang, et al.) to see if the cleavage assay actually works. If it does not work with the paper sequences, then the experiment procedures would need to be modified or should try with another type of DNAzyme.
2. (2) As shown in the Nanodrop results during the 1st cycle, the purity and quality of DNA might be a problem in the experiments. Therefore, we can try using synthetic DNA from another company (ex. IDT) to problematize the experiments. If cleavage assay does not work with newly ordered DNA from IDT, then the experiment procedures would need to be modified or should try with another type of DNAzyme.

3rd cycle

*1. Imagine (brainstorming) & Design*
    Based on the DNAzyme and gene substrate sequences that were provided in the reference paper of F-8 DNAzyme (Wang, Zhang, et al.), we added additional sequences to the left and right arms of the sequence in the paper, to increase the length of synthetic DNA. We designed it so that each single stranded DNA will not self-hybridize, and that it would hybridize with each other instead. We have used a program to ensure that the DNAzyme\_Paper and Gene\_Paper strands would hybridize to each other well. The following shows the DNAzyme\_Paper and the corresponding Gene\_Paper sequence. The highlighted sequence in yellow are the base pairs that were added by us additionally (the black letters are the original sequences that were provided by the reference paper).

| Name | Sequence (5’→3’) |
| --- | --- |
| Gene\_Paper | TTTTGGGGTTTGGGTTGGTGTGCCGATCCATACTGCGGAACACTTGTTGGTTTGGGTTTTGGGG |
| DNAzyme\_Paper | CCCCAAAACCCAAACCAACAAGTGTTCCGTGGATGGAGCAATAGTCTCCCGGGTCCGTATGGATCGGCACACCAACCCAAACCCCAAAA |

    In addition, we recognized the need to see how the cleavage activity changes as the concentration of the reaction buffer changes. We have found that the Cu2+ ions are especially important in catalyzing the DNAzyme activity. In this context, we hypothesized that increasing the concentration of the reaction buffer (thus, increasing the concentration of Cu2+ ions) would enhance the cleavage activity, which would lead to more of the gene substrate to be cleaved. If this tendency is shown in PAGE results, this would ensure that the cleavage activity is actually exercised by the DNAzymes.

*2. Test*
The synthetic DNA was ordered from IDT.

We designed the cleavage assay experiments based on the protocol provided by Wang, Zhang, et al. The experiment procedure is the same as in the 1st and 2nd cycle. The controls are the same as in the 1st cycle.

| Sample | P-1 | P-2 | P-3 | P-4 |
| --- | --- | --- | --- | --- |
| 10 µM DNAzyme | 2 µL | - | 2 µL | 2 µL |
| 1 µM Substrate | - | 2 µL | 2 µL | 2 µL |
| Ions (2x reaction buffer) | 2 µL | 2 µL | - | 2 µL |
| 100mM HEPES (pH 7.4) | - | - | 2 µL | - |
| Milli-Q water | 16 µL | 16 µL | 14 µL | 14 µL |

    One difference from the cleavage assay in the 1st cycle is that we tried at different concentrations of the reaction buffer. The standard concentration is 2x reaction buffer (Wang, Zhang, et al.), but we also tested at 1x, 3x, 4x, and 5x.

    To visualize the results, native PAGE was performed under 90V. To optimize the conditions of PAGE, it was performed at different polyacrylamide gel concentrations (12% and 15%) and at different gel running times (90 minutes and 120 minutes).

    3 rounds of experiments were performed with the DNAzyme-substrate Paper sequences to show that the experiment produced consistent results.

*3. Learn & Improve*

![igem_yonsei](https://static.igem.org/mediawiki/2021/6/6e/T--Yonsei_Korea--success_5.png)

    All 3 rounds of experiments showed consistent results: the band patterns were the same, regardless of the reaction buffer concentrations (1x/2x/3x/4x/5x) and the polyacrylamide gel concentrations (12% or 15%). In addition, it was possible to observe the decrease in the band intensity in sample 4, as the concentration of the reaction buffer increased. This ensures not only that Cu2+ ions are important in F-8 DNAzyme activity, but also ensures that the DNAzyme cleavage activity is working.

    Sample 3 bands show that hybridization between the DNAzyme and gene substrate occurs, since the bands are appearing near the top. Also, there is a shift in the sample 4 bands, when it is compared with sample 3. As the gene substrate is cleaved, the shape of the DNAzyme-substrate complex will change, leading to the shift in the bands.

    Overall, we learned that cleavage activity is working with the sequences from the reference paper (Wang, Zhang, et al.), meaning that the cleavage reaction with the F-8 DNAzymes and our current experiment methods are actually working. This suggests that the quality of the DNA might have been interfering with the cleavage assay. For our next experiments, we decided to perform the same experiment with the DNAzyme-substrate A sequences, but with the synthetic DNA that were synthesized by IDT.

*4. Design & Test*
    The synthetic DNA was ordered from IDT. Also, the same experiment conditions and procedures with the DNAzyme-substrate Paper were used. To visualize the results, native PAGE (12% and 15%) was performed under 90V for 120 minutes.

*5. Learn & Improve*

![igem_yonsei](https://static.igem.org/mediawiki/2021/f/f2/T--Yonsei_Korea--success_6.png)

    Regardless of the reaction buffer concentrations (1x/2x/5x) and the polyacrylamide gel concentrations (12% or 15%), the band patterns were the same. In other words, consistent results were obtained. In addition, it was possible to observe the decrease in the band intensity in sample 4, as the concentration of the reaction buffer increased. The cleavage reaction for sample 4 was possible compared to sample 3, because the reaction buffer contained Cu2+ and Mn2+, which are the most important ions. Without them, DNAzyme cannot catalyze the cleavage of its substrate, as seen in sample 3.

    Sample 3 bands show that the hybridization between the DNAzyme and gene substrate occurs, since one additional band is appearing near the top: (a). In sample 4 bands, there is a decrease in band intensity at the (a) position. In addition, there is another band appearing slightly below: (b).

1. ● Band (a) sample 3 vs sample 4: DNAzyme exercised cleavage activity, so there is less amount of uncleaved DNAzyme-gene A complex.
2. ● Band (b): Due to cleavage at CTGC, the overall shape of the complex might have changed, possibly forming secondary DNA structure. During its cleavage, it would have released the 4bp sequence, while the rest of the gene substrate is still being bound to the DNAzyme arms. The shift in the band from band II to band III clearly suggests that the gene substrate was cleaved by the DNAzyme.

    The following is a diagram of structures at band (a) and (b):

1. ● Green: DNAzyme
2. ● Yellow: Left-binding arm of the target gene
3. ● Red: CTGC sequence - sequence where DNAzyme specifically recognizes the target gene
4. ● Orange: Right-binding arm of target gene

![igem_yonsei](https://static.igem.org/mediawiki/2021/f/f2/T--Yonsei_Korea--success_7.png)

    For improving our research for the 4th cycle, we need to consider the following:

1. (1) Confirmation of the DNAzyme specificity: proof that the DNAzyme exercises cleavage activity based on specific base pairing is needed.
2. (2) Confirmation that DNAzyme cleavage activity is specifically catalyzed by Cu2+ ions

4th cycle

*1. Research & Imagine (brainstorming)*
    In the 4th cycle, we aimed to confirm that our detection system was efficient and what determines the cleavage activity of the DNAzyme. According to Wang, Zhang, et al., Cu2+ seemed critical in the DNAzyme activity, although they have mentioned Mn2+ as well. Up to the 3rd cycle, we have proved that our DNAzyme works under the reaction buffer, but we have not tested which ions are most significant in catalytic activity. Therefore, we planned to test the DNAzyme cleavage activity in the absence of Cu2+ ions.

    Second, we wanted to confirm that our system is an effective system: the cleavage activity of DNAzyme should be proportional to the amount of gene (substrate) present. Therefore, we planned to see how the DNAzyme cleavage activity varies as the concentration of the gene (substrate) changes.

    Lastly, we decided to use denaturing PAGE (urea PAGE) this time, in order to separate molecules based solely on the molecular weight, as urea unfolds possible secondary structures and destroys hybridization between strands.

*2. Design*
    Based on the research & imagine (brainstorming) stage, we have decided to make a 2x reaction buffer without Cu2+ ions. The composition of other ingredients and ions are the same, so it will be possible to see whether or not Cu2+ is essential for cleavage activity. In addition, we made different concentrations of gene (substrate), while keeping the concentration of DNAzyme constant. We designed our experiment to test at 4 different ratios - DNAzyme : Gene (substrate) = 1 : 0.1, 1 : 0.25, 1 : 0.5, 1: 1. Since 10µM DNAzyme is used, the concentration of the substrate would be: 1µM, 2.5µM, 5µM, and 10µM.

*3. Test*
    The synthetic DNA was ordered from IDT. The experiment procedure is the same as in 1st, 2nd, and 3rd cycle, but with different reaction conditions and controls.

| Ladder (Substrate concentration) | A-1 (1 µM) | A-2 (1 µM) | A-3 (1 µM) | A-4 (1 µM) | A-5 (2.5 µM) | A-6 (5 µM) | A-7 (10 µM) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 10 µM DNAzyme | 2 µL | - | 2 µL | 2 µL | 2 µL | 2 µL | 2 µL |
| Substrate | - | 2 µL | 2 µL | 2 µL | 2 µL | 2 µL | 2 µL |
| Ions (2x reaction buffer) | 2 µL | 2 µL | - | 2 µL | 2 µL | 2 µL | 2 µL |
| 2x without CuCl2 | - | - | 2 µL | - | - | - | - |
| Milli-Q water | 16 µL | 16 µL | 14 µL | 14 µL | 14 µL | 14 µL | 14 µL |

    To visualize the results, denaturing (urea) PAGE was performed. 8% urea PAGE gel was made, and it was run at 200V for 40 minutes.

*4. Learn & Improve*

![igem_yonsei](https://static.igem.org/mediawiki/2021/c/c9/T--Yonsei_Korea--success_8.png)

    Samples A-4 to A-7 had all the needed reagents. In contrast, the A-3 contained all the required reagents except for Cu2+ ions. We confirmed that DNAzyme must have the Cu2+ ions to cleave the substrate because we did not detect the middle band in Lane 5, which were observed in remaining lanes.

    Samples A-4 to A-7 had an increasing concentration of the target substrate, while that of DNAzyme remained unchanged at 10 uM. The 1:1 ratio between DNAzyme and substrate (10uM : 10uM) was the most effective reaction since we observed a shrunken band size of DNAzyme compared to other samples. This indicated that DNAzyme cleaved the available substrate fully, leaving only unreacted DNAzymes.

    For further analysis, we used Fiji, an open source image analysis software, to quantify the band intensity. Here, the PAGE results from the 3rd cycle were analyzed as well. The band intensity was measured to quantify the amount of DNA present in each band. In case of PAGE results, the picture was adjusted to an 8-bit image because the original background was blue. Then, the quantification process was conducted as:

1. 1) Measure the mean light intensity of the background and each band.
2. 2) Subtract the background value from the value of each band to calculate the net band intensity.

    After the net band intensity was calculated, we analyzed based on the results. A detailed description of these results can be found in the [Results](https://2021.igem.org/Team%3AYonsei_Korea/Results) section.

    Through this, we were able to conclude the following:

1. ● It is clear that the DNAzyme participates in the cleavage reaction, and this amount is proportional to the concentration of the ions that catalyze its cleavage activity.
2. ● DNAzyme cleavage activity is present only in the presence of certain ions. Without it, DNAzyme and substrate only hybridize: no cleavage activity is observable.
3. ● Cu2+ is specifically required for the DNAzyme cleavage activity, since it highly increases the cleavage efficiency. Without Cu2+, cleavage activity is hardly observable.
4. ● DNAzyme cleavage activity is proportional to the amount of DNA (substrate present), which makes the detection system based on the DNAzyme cleavage assay effective and efficient.

Conclusion of Phase 1

    Based on synthetic biology, we were able to prove that the DNAzyme designed based on the target gene sequence was successful in specifically detecting the target DNA by cleaving the DNA. This cleavage activity is efficient under the presence of Cu2+ ions, and is proportional to the amount of target DNA present. We were able to achieve this based on simplicity, which makes our system highly advantageous. Without using any specific enzyme, expensive purification, instrumentation, and advanced technology, detection was possible based on solely synthetic DNA.

PHASE TWO

Phase 2: Development of a colorimetric system to visualize the specific detection via color change

1st cycle

*1. Research*
    After making the decision to include a nano component in our research, we began to heavily research different detection mechanisms or visual readouts that aligned with our initial concept design. From this we made a list of two main components including ease of use or simplicity and clear visual color change that is noticeable with the naked eye. Out of our options, gold nanoparticles were the most promising step as they possess many benefits and advantages such as being non-toxic, cheap and can be easily functionalized.

    Gold nanoparticles are also sensitive to the distance between each of their particles, so if something triggers their aggregation, then their surface plasmon resonance will become coupled and shift their absorbance. Gold nanoparticles that are dispersed show a red color while aggregated particles show a purple or blue color (Zagorovsky, Chan, et al).

    We read many research papers and review articles on gold nanoparticles and how they can be used as a colorimetric detection tool for specific DNA sequences or for pathogens. After reviewing many different papers with our nano team, we were able to finalize a narrow focus and method to functionalize specific DNA sequences onto gold nanoparticles which could be used to detect the complementary sequences and aggregate to form a color change.

*2. Imagine (brainstorming)*
    Once we narrowed down our focus, we continued to read and review more papers on specific methods and procedures to functionalize DNA sequences onto gold nanoparticles as well as think about how our overall detection system will work. In essence, we wanted to make it very simple to the point where it was adding A to B and seeing a color change with the naked eye. This was to cover our key point which was simplicity. Moreover, with simplicity comes a lower cost in most cases, so we wanted to also develop something that would be cheap and easily accessible to people.

    Therefore, through brainstorming with our nano team, we established the concept to our mechanism or detection system: we wanted to attach specific DNA sequences to the nanoparticles which are complementary to our target gene that we can obtain from Magnaporthe oryzae. After this, we would add the actual target gene into the gold nanoparticle solution in order for it to hybridize or complement back that which was functionalized on the nanoparticles and change the aggregation/dispersion properties of them, thus causing a color change. Nevertheless, here came one challenge which was to specify exactly when and how the nanoparticles would aggregate, making sure they are specific to our target gene as well as stable in solution. We were able to find one protocol as well as a design concept that matched exactly what we were aiming for which will be covered next in the Design section.

*3. Design*
    We chose to functionalize the 20 base pairs from the 3’ and 5’ end of our target gene on two pools of gold nanoparticle sets, respectively. With this, we aim to create a square-like aggregate structure when these functionalized gold nanoparticles come in contact with our target gene. In order for them to aggregate, there must be a space between each DNA strand functionalized on the nanoparticles once hybridized with the complementary target gene. This ensures that they will bind specifically to our target gene. For example, if our target gene is 3’-AAAABBAAABBAAAA-5’, then we functionalize 5’-BBBBA-3’ on the first set of nanoparticles and 5’-ABBBB-3’ on the set of second nanoparticles. What will be the space or blank here between the strands is the middle part of the target gene. Nevertheless, when the target gene is added to this solution containing both these functionalized gold nanoparticles, it will hybridize accordingly, forming a cross-linked, square-like aggregate structure, turning the solution purple as the particles are not freely dispersed anymore.

![igem_yonsei](https://static.igem.org/mediawiki/2021/0/03/T--Yonsei_Korea--implementation_3.png)

    Here we can see the target gene and thiolated sequences (which are the DNA strands functionalized onto our gold nanoparticles) we chose:

    In addition, in order for the DNAzymeA to be activated, important ions such as Cu2+ is essential. Therefore, with the addition of Cu2+, the target Gene A will be cleaved as below:

Target Gene
    1.  TG: 5’-acggccagtgccggcgacagctctagcaaccccactggctcgg-3’

Thiolated Sequences (complementary to target gene)
    1.  TS1: 3’-tgccggtcacggccgctgtc-5’
    2.  TS2: 3’-atcgttggggtgaccgagcc-5’

![igem_yonsei](https://static.igem.org/mediawiki/2021/f/ff/T--Yonsei_Korea--concept_5.png)

    The target gene is one of the products of the cleavage reaction using the DNAzymes earlier in the lab. There are three products, but we chose to use this one above as our target gene for our detection system. In essence, if this were to be developed into one system, the goal is that if the solution changes to purple then our target gene has been detected which could only mean that the DNAzyme cleavage reaction was successful. Although here we do not include the DNAzyme and its cleaving reaction with our gold nanoparticles solution, it is the ultimate goal. Here we simply wanted to check that our nanoparticles react to our target gene and nail down the cross-linking aggregation that occurs as a result of the complementary DNA hybridization. The ultimate goal surely is to combine both the synthetic biology system with the nanoparticles system into one to make an integrated detection tool; however, that is planned for future prospects of our project.

*4. Test*
    The synthetic and thiolated DNA was ordered from IDT Korea. This nano component was divided into two parts: functionalization of gold nanoparticles and hybridization with the target gene. We conducted the functionalization of gold nanoparticles with our DNA sequences based on the protocol adapted from Hill, et al. The hybridization part is also adapted from the same protocol.

    For the first part, to functionalize our gold nanoparticles with the thiolated sequences, we used the salt-aging method whereby a salting buffer is added over the course of two days. Firstly, the thiolated DNA sequences (TS1: 3’-tgccggtcacggccgctgtc-5’ and TS2: 3’-atcgttggggtgaccgagcc-5’) were re-hydrated with PBS (pH8), wrapped in foil and kept in an oven at 50 degrees celsius for around 35 minutes. Our protocol stated to let them stand at room temperature for 2-3h; however, we chose to speed up the process by using an oven. After this, the DNA was passed through a Nap-5 column and added to a solution of gold nanoparticles (30 nm). In order to quantify the amount of DNA that passed through the Nap5-columns and into our samples that were aliquoted every 3-4 drops, we used a nanodrop UV-Vis spectrometer to view the samples with strong absorbance at 260 nm which is the wavelength at which DNA absorbs light.

    At this point we have 2 samples each for TS1 and TS2, so 4 samples in total (A1-4). Phosphate adjustment buffers, surfactant solutions and salting buffers were then added to the solution where the salting buffer was added lastly over the course of 2 days every 8 hours.

| Samples | A1 | A2 | A3 | A4 |
| --- | --- | --- | --- | --- |
| Total amount of DNA obtained | 560 µL | 650 µL | 560 µL | 650 µL |
| PBS solution (pH 8) | 100 µL | 100 µL | 100 µL | 100 µL |
| Gold nanoparticles (30 nm) | 1 mL | 1 mL | 1 mL | 1 mL |
| Phosphate adjustment buffer (100 mM) | 156 µL | 165 µL | 159 µL | 148 µL |
| Surfactant solution (10% SDS (wt/v)) | 17.16 µL | 18.15 µL | 17.49 µL | 16.28 µL |
| Salting buffer (2M NaCl) | 43 µL | 45.5 µL | 43.8 µL | 40.8 µL |

    For the next step including the hybridization of our functionalized gold nanoparticles with our target gene, they are still in trial.

    Due to the wiki freeze deadline, we are still in the process of the next phase to hybradize our functionalized gold nanoparticles with our target gene and observe a color change from red to purple. We shall present these results in time for the Giant Jamboree.

*5. Learn & Improve*

Conclusion of Phase 2

    As we are still conducting the nanoparticles experiments in the lab due to setbacks in accessing the adequate lab spaces. Nevertheless, we have completed the first part which includes functionalizing the gold nanoparticles with our thiolated DNA sequences. The next step which is in progress is to complete the successful hybridization of our target gene to our nanoparticles.

FUTURE PROSPECT

*1. Working with real rice blast infected leaves and M. oryzae (in vivo)*
    The Ministry of Food and Agriculture in Korea has set strict restrictions on experimenting with M. oryzae in labs. Therefore, it was not possible for us to test our system in vivo: we were limited to test it as a cell-free system. We have contacted research institutions in the Ministry of Food and Agriculture and university labs that study the rice blast disease on whether we would be able to experiment in those labs under supervision or if we could obtain the extracted M. oryzae genome. However, it was not possible to get it either way, due to COVID-19 and administrative restrictions. To further develop our detection system into a tool or a kit, working with real rice blast infected leaves and M. oryzae will be important to show that our system is practical to use in the real world.

*2. Integration of phase 1 & 2 into developing a rice blast detection kit*
Our ultimate goal is to develop a fully functioning kit or device that farmers could easily use to detect rice blast disease early in their crops. Therefore, we are planning to continue our research by integrating the synthetic biology part (phase 1) with the nanoscience and technology part (phase 2) into a simple detection kit/device.

REFERENCES

1. 1.
   [Chadha, S., & Gopalakrishna, T. (2006). Detection of Magnaporthe grisea in infested rice seeds using polymerase chain reaction. Journal of applied microbiology, 100(5), 1147–1153.](https://doi.org/10.1111/j.1365-2672.2006.02920.x)
2. 2.
   [Chen, W-L., Lin, Y-B., Ng, F. L., Liu, C-Y., & Lin, Y-W. (2020). RiceTalk: Rice Blast Detection Using Internet of Things and Artificial Intelligence Technologies. IEEE Internet of Things Journal, 7(2), 1001-1010. [8871173].](https://doi.org/10.1109/JIOT.2019.2947624)
3. 3.
   [Li, L., Zhang, S. Y., & Zhang, C. Q. (2019). Establishment of a Rapid Detection Method for Rice Blast Fungus Based on One-Step Loop-Mediated Isothermal Amplification (LAMP). Plant disease, 103(8), 1967–1973.](https://doi.org/10.1094/PDIS-11-18-1964-RE)
4. 4.
   [Magar, P. B., Acharya, B., & Pandey, B. (2015). Use of Chemical Fungicides for the Management of Rice Blast (Pyricularia Grisea) Disease at Jyotinagar, Chitwan, Nepal. International Journal of Applied Sciences and Biotechnology, 3(3), 474–478.](https://doi.org/10.3126/ijasbt.v3i3.13287)
5. 5.
   [Selisana, S. M., Yanoria, M. J., Quime, B., Chaipanya, C., Lu, G., Opulencia, R., Wang, G. L., Mitchell, T., Correll, J., Talbot, N. J., Leung, H., & Zhou, B. (2017). Avirulence (AVR) Gene-Based Diagnosis Complements Existing Pathogen Surveillance Tools for Effective Deployment of Resistance (R) Genes Against Rice Blast Disease. Phytopathology, 107(6), 711–720.](https://doi.org/10.1094/PHYTO-12-16-0451-R)
6. 6.
   [Sun, G., Liu, J., Li, G., Zhang, X., Chen, T., Chen, J., Zhang, H., Wang, D., Sun, F., & Pan, H. (2015). Quick and Accurate Detection and Quantification of Magnaporthe oryzae in Rice Using Real-Time Quantitative Polymerase Chain Reaction. Plant disease, 99(2), 219–224.](https://doi.org/10.1094/PDIS-05-14-0485-RE)
7. 7.
   [Wang, M., Zhang, H., Zhang, W., Zhao, Y., Yasmeen, A., Zhou, L., Yu, X., & Tang, Z. (2014). In vitro selection of DNA-cleaving deoxyribozyme with site-specific thymidine excision activity. Nucleic acids research, 42(14), 9262–9269.](https://doi.org/10.1093/nar/gku592)
8. 8.
   [Zagorovsky, K. and Chan, W.C.W. (2013), A Plasmonic DNAzyme Strategy for Point-of-Care Genetic Detection of Infectious Pathogens. Angew. Chem. Int. Ed., 52: 3168-3171.](https://doi.org/10.1002/anie.201208715)
9. 9.
   Hill, Haley D., and Chad A. Mirkin. "The bio-barcode assay for the detection of protein and nucleic acid targets using DTT-induced ligand exchange." Nature protocols 1.1 (2006): 324-336.

YONSEI\_KOREA 2021
![](https://static.igem.org/mediawiki/2021/b/b7/T--Yonsei_Korea--main_logo_1.png)

Yonsei\_Korea is a convergent team of students based in Seoul, South Korea striving to create meaningful change utilizing synthtic biology in the iGEM 2021 Competition.

* Engineering Success
* Overview
* Phase 1
* Phase 1\_1st Cycle
* Phase 1\_2nd Cycle
* Phase 1\_3rd Cycle
* Phase 1\_4th Cycle
* Phase 1\_Conclusion
* Phase 2
* Phase 2\_1st Cycle
* Phase 2\_Conclusion
* Future Prospect
* References

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Yonsei Korea/Experiments

IGEM\_YONSEI

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In the Laboratory:

The **Science**
Behind It All

EXPERIMENTS

DNAzyme CLEAVAGE ASSAY

PART 1: REACTION BUFFER PREPARATION

1. *A. Materials*
   1. : 1M HEPES (pH 7.4), KCl, NaCl, MgCl2⋅6H2O, MnCl2⋅4H2O, CuCl2⋅2H2O, Milli-Q water, 15mL tube, vortex, weighing paper, plastic spoons, scale, labeling tape
2. *B. Procedure*
   1. : Prepare 10000 µL of 5x reaction buffer based on the calculations below.
      1. - Since the mass of CuCl2 2H2O to make 0.25mM CuCl2 2H2O for 5x reaction buffer is too small, make a separate 100mM CuCl2 2H2O solution first. Use this as a stock solution to prepare 0.25mM CuCl2 2H2O for the 5x reaction buffer.
      2. - When preparing the solutions, make sure to label each solution.
      3. - Vortex the solution until the solids are all dissolved.

|  | Volume needed (μL) | Stock (M) | Mass needed (g) | Molar mass (g/gmol) |
| --- | --- | --- | --- | --- |
| Solution 1: 5x Reaction Buffer | | | | |
| 250mM HEPES (pH 7.4) | 2500 | 1 |  |  |
| 2M NaCl |  |  | 1.169 | 58.44 |
| 0.5M KCl |  |  | 0.373 | 74.55 |
| 50mM MgCl2 6H2O |  |  | 0.102 | 203.3 |
| 37.5mM MnCl2 4H2O |  |  | 0.074 | 197.7 |
| 0.25mM CuCl2 2H2O | 25 | 0.1 |  |  |
| Buffer total volume | 10000 (= 10mL) | | | |
|  | | | | |
| Solution 2: 100mM CuCl2 2H2O solution | | | | |
| CuCl2 2H2O |  |  | 0.102 | 170.48 |
| Total volume | 6000 (= 6mL) | | | |

PART 2: DNAzyme CLEAVAGE REACTION

1. *A. Materials*
   1. : DNAzymeA, GeneA, buffer, Milli-Q water, DEPC H2O, test tube, vortex, 1.5mL microcentrifuge tube, 1M HEPES (pH 7.4), pipettes, pipette tips, heat block
2. *B. Procedure*
   1. \*\*Note: keep the samples in ice.
      1. 1. Preparation of 10 µM DNAzyme A: Dilute your stock sample depending on the stock concentration. Use DEPC H2O in dilution.
      2. 2. Preparation of 1µM Substrate (Gene A): Dilute your stock sample depending on the stock concentration. Use DEPC H2O in dilution.
      3. 3. Dilute 5x reaction buffer (prepared from Part 1 above) to make 2x reaction buffer. Use Milli Q water to dilute the reaction buffer.
      4. 4. In a single 1.5mL microcentrifuge tube, insert the following:

         | Ingredient | Volume (µL) |
         | --- | --- |
         | 10 µM DNAzyme | 2 |
         | 1 µM Substrate | 2 |
         | 2x reaction buffer | 2 |
         | Milli-Q water | 14 |
         | Total volume | 20 |
      5. 5. Heat the DNAzyme-substrate mixture at 90°C for 5 minutes using a water bath.
      6. 6. Cool the mixture at 25°C room temperature for 10-15 minutes.
      7. 7. Incubate at 37℃ for 16 hours (overnight).

PART 3: STOPPING THE CLEAVAGE REACTION WITH EDTAN

1. *A. Materials*
   1. EDTA, 20µl pipette, 20µl pipette tips
2. *B. Procedure*
   1. 1. Take out the samples from the 37°C incubator after 16 hours.
   2. 2. Using a 20µl pipette, take 2µl of EDTA and insert it into all of the samples to stop the DNAzyme cleavage activity.
   3. 3. Store the samples and solutions in the fridge.

AGAROSE GEL ELECTROPHORESIS

1. *A. Materials*
   1. Agarose powder, 0.5x TAE buffer, comb, gel tray, 6x loading dye, 1kb DNA ladder plastic plate, scale, spoon, glass bottle, plastic protective holders, graduated cylinder, 20µl pipette, 20µl pipette tips, ice bucket, vortex mixer, spin down machine, power supply (for applying voltage to the gel electrophoresis), rack, labeling tape, microwave, gel box, UV light lamp.
2. *B. Procedure*
   1. 1. Take the gel tray and the comb, and assemble the gel cassette.
      \*\*Note: choose the comb that has the desired number of wells, depending on the number of samples.
   2. 2. Take the dried plastic plate and zero the scale.
   3. 3. Making 1% agarose gel:
      1. a. Measure 0.4g of agarose powder with the plastic spoon.
      2. b. Put the measured agarose powder into the glass bottle.
      3. c. Measure 40mL of 0.5x TAE buffer using the 100mL graduated cylinder.
      4. d. Pour the TAE buffer into the glass bottle, which has the agarose powder inside.
         \*\*Note: don’t close the glass bottle lid tightly, since it might burst during the microwave.
   4. 4. Microwave the mixture for 2 minutes and 30 seconds.
   5. 5. Use the protective glove to take out the heated glass bottle out of the microwave.
   6. 6. Cool the agarose mixture on the bench until it is possible to touch it with your bare hands for 3 seconds.
   7. 7. Pour the cooled agarose mixture into the tray that has been set up in step #1.
   8. 8. Leave it for 30 minutes, so that the gel will solidify.
   9. 9. While the gel is being solidified, prepare your loading samples:
      1. a. Take the 6x loading dye out of the refrigerator and place it in the ice.
      2. b. Take 4.5µl of 6x loading dye and put it into each sample (the volume of each sample is 22µl).
         \*\*Note 1: after inserting the 6x loading dye into our samples, we have to change the pipette tip.
         \*\*Note 2: the volume of the 6x loading dye will depend on the sample volume.
      3. c. Vortex and spin down the mixture until it is fully mixed.
         \*\*Note: before loading the mixture into the wells, vortex and spin down once more.
      4. d. After using the 6x loading dye, make sure to put it back into the refrigerator as soon as possible, since it is sensitive to temperature and light.
   10. 10. Once the gel is solidified, take out the comb. Take out the gel and place it inside the gel box. Make sure the gel is fully covered with the 0.5x TAE buffer.
   11. 11. Load 3ul of the 1kb DNA ladder inside the 1st lane. Leave the 2nd lane empty.
   12. 12. Load the prepared samples into the wells, starting from the 3rd lane.
       \*\*Note: make sure to keep the order of the samples, so that we are not confused.
   13. 13. Close the lid and connect the power supply.
   14. 14. Set the power supply to 30 minutes and 135V.
   15. 15. Visualize the results, using the UV light lamp:
       1. a. When the gel electrophoresis is complete, turn off the power.
       2. b. Use the transparent, plastic tray to carry your gel.
       3. c. Turn on the UV light visualization machine.
       4. d. Set the machine to “Nucleic Acid Gel”
   16. 16. After viewing the results, clean up the machine:
       1. a. After taking out the gel, clean the machine with 4% EtOH and Kimtech wipers (tissue).
       2. b. Turn off the machine.

PAGE

1. *A. Equipment*
   1. Glass plates (small and tall plates) with integrated spacers, casting frame, casting stand, plastic combs, electrophoresis chamber, power supply, lid, serological pipette, an automatic pipette aid.
2. *A. Reagents*
   1. 40% acrylamide gel, 10% APS (Ammonium Persulfate), TEMED, 5x TBE (Tris Boric EDTA) buffer, MilliQ water, samples, 10 bp DNA ladder.
3. *A. Procedure*
   1. 1. Assembling the gel casting apparatus:
      1. 1.1. Take the short and tall glass plates and make sure they are dry. If not, use a lint free wipes to dry them.
      2. 1.2. Take the casting frame and place it on the bench with its clamps open (perpendicular to the frame) and facing you.
      3. 1.3. Insert the glass plates into the frame. Place the taller glass plate with its spacer and UP arrows up and the short glass plate in front of the casting frame. Make sure that the botted edges of the two plates MUST be even!
      4. 1.4. Secure the glass plates in the casting frame by pushing the two gates of the frame out to the sides.
      5. 1.5. Place the casting frame with glass plates into the casting stand and clamp them.
      6. 1.6. Check to see if the apparatus is fully assembled by pipetting a small amount of D.W. If the glass plates do not leak, you are ready to prepare the gel. Pour the D.W. out by holding the entire casting platform over a sink. Use the lint free tissues to absorb any residual D.W.
   2. 2. Preparing the resolving gel:
      1. 2.1. Take 2 new 15 mL tubes and label them as 15% and 10%.
      2. 2.2. Add all the required reagents according to the given information on the table:

         | 10% Native PAGE | Total volume - 8 mL |
         | --- | --- |
         | 40% acrylamide gel | 2.0 mL |
         | 10% APS | 80 uL |
         | TEMED | 8 uL |
         | Milli Q water | 5.2 mL |
         | 5x TAE | 0.8 mL |

         | 15% Native PAGE | Total volume - 8 mL |
         | --- | --- |
         | 40% acrylamide gel | 2.3 mL |
         | 10% APS | 80 uL |
         | TEMED | 8 uL |
         | Milli Q water | 4.8 mL |
         | 5x TAE | 0.8 mL |
      3. 2.3. Immediately mix the reagents by vortexing.
      4. 2.4. Pour the gel immediately using the serological pipette and an automatic pipette aid between the two glass plates.
      5. 2.5. Immediately insert the plastic comb (15 wells) into the gel.
      6. 2.6. Leave the gel for 15 min at room temperature to completely harden.
   3. 3. Running the gel:
      1. 3.1. Once the gel is hardened, place it inside the electrophoresis chamber.
      2. 3.2. Make 0.5x TBE buffer by mixing 950 mL of Milli Q water and 50 mL of 10x TBE buffer.
      3. 3.3. Add 0.5x TBE buffer to completely fill the inner chamber between the glass plates.
      4. 3.4. Add 0.5x TBE buffer to the chamber until it matches the 2 gel sign written on the chamber wall.
      5. 3.5. Carefully remove the comb and load the samples into the wells of the gel.
      6. 3.6. Close the lid and connect the cables to the power supply. Make sure to connect the black and red cables to the corresponding colours on the supply.
      7. 3.7. Run the gel at 90V for 2 hours.
   4. 4. Visualising the results:
      1. 4.1. Once the running process is complete, turn off the power and remove the lid carefully.
      2. 4.2. Prepare the tray and pour a small amount of D.W in it.
      3. 4.3. Take the gel cassette carefully and discard the buffer inside the inner chamber.
      4. 4.4. Release the clamps and take the glass plates out.
      5. 4.5. Place the glass plates onto the tray and carefully remove the gel.
      6. 4.6. Visualise the results using the UV machine.

DENATURING PAGE (UREA PAGE)

1. *A. Equipment*
   1. Glass plates (small and tall plates) with integrated spacers, casting frame, casting stand, plastic combs, electrophoresis chamber, power supply, lid, serological pipette, an automatic pipette aid.
2. *A. Reagents*
   1. 10% APS (Ammonium Persulfate), TEMED, 5x TBE buffer, formamide, samples.
3. *A. Procedure*
   1. 1. Assembling the gel casting apparatus:
      1. 1.1. Take the short and tall glass plates and make sure they are dry. If not, use a lint free wipes to dry them.
      2. 1.2. Take the casting frame and place it on the bench with its clamps open (perpendicular to the frame) and facing you.
      3. 1.3. Insert the glass plates into the frame. Place the taller glass plate with its spacer and UP arrows up and the short glass plate in front of the casting frame. Make sure that the botted edges of the two plates MUST be even!
      4. 1.4. Secure the glass plates in the casting frame by pushing the two gates of the frame out to the sides.
      5. 1.5. Place the casting frame with glass plates into the casting stand and clamp them.
      6. 1.6. Check to see if the apparatus is fully assembled by pipetting a small amount of D.W. If the glass plates do not leak, you are ready to prepare the gel. Pour the D.W. out by holding the entire casting platform over a sink. Use the lint free tissues to absorb any residual D.W.
   2. 2. Preparing the gel:
      1. 2.1. Take a new 15 mL tube.
      2. 2.2. Add all the required reagents according to the given information on the table:

         | 8% Urea PAGE gel | Total volume - 10 mL |
         | --- | --- |
         | 40% acrylamide gel | 3.2 mL |
         | Urea | 5.8 mL |
         | 5x TBE buffer | 1.2 mL |
         | 10% APS | 100 uL |
         | TEMED | 10 uL |
      3. 2.3. Immediately mix the reagents by vortexing.
      4. 2.4. Pour the gel immediately using the serological pipette and an automatic pipette aid between the two glass plates.
      5. 2.5. Immediately insert the plastic comb (15 wells) into the gel.
      6. 2.6. Leave the gel for 15 min at room temperature to completely harden.
   3. 3. Preparing the resolving gel:
      1. 3.1. Take the samples out of the storage at -20°C and melt them on ice.
      2. 3.2. Take the formamide and keep it on ice.
      3. 3.3. Set the heat block to heating mode at 90°C.
      4. 3.4. Take new 1,5 mL microcentrifuge tubes and label them according to the experiment condition.
      5. 3.5. Add 1:1 ratio of the samples and formamide to the labeled tubes. For instance, you can add 10 uL of the sample and 10 uL of formamide.
      6. 3.6. Gently vortex the mixture and place it on a heat block at 90°C for 20 minutes to denature dsDNAs.
      7. 3.7. After 20 minutes, take the samples. Cool the samples by using the bench mini centrifuge to spin for 1 minute.
   4. 4. Running the gel:
      1. 4.1. Once the gel is hardened, place it inside the electrophoresis chamber.
      2. 4.2. Make 0.5x TBE buffer by mixing 950 mL of Milli Q water and 50 mL of 10x TBE buffer.
      3. 4.3. Add 0.5x TBE buffer to completely fill the inner chamber between the glass plates.
      4. 4.4. Add 0.5x TBE buffer to the chamber until it matches the 2 gel sign written on the chamber wall.
      5. 4.5. Carefully remove the comb and load the samples into the wells of the gel.
      6. 4.6. Close the lid and connect the cables to the power supply. Make sure to connect the black and red cables to the corresponding colours on the supply.
      7. 4.7. Run the gel at 200V for 40 minutes.
   5. 5. Visualising the results:
      1. 5.1. Once the running process is complete, turn off the power and remove the lid carefully.
      2. 5.2. Prepare the tray and pour a small amount of D.W in it.
      3. 5.3. Take the gel cassette carefully and discard the buffer inside the inner chamber.
      4. 5.4. Release the clamps and take the glass plates out.
      5. 5.5. Place the glass plates onto the tray and carefully remove the gel.
      6. 5.6. Visualise the results using the UV machine.

DNA FUNCTIONALIZED GOLD NANOPARTICLES
(SALT-AGING METHOD)

1. *A. Equipment*
   1. Vortex machine, orbital shaker, oven
2. *A. Materials*
   1. Thiolated Sequence 1 (TS1): 3’-tgc cgg tca cgg ccg ctg tc-5’ and thiolated Sequence 2 (TS2): 3’-atc gtt ggg gtg acc gag cc-5’, PBS (pH 8), Na2HPO4, NaH2PO4, NaCl, 1.5mL microcentrifuge tubes, 15mL tube, nap5 columns, labelling tape, metal spatula, weighing paper, purified water

      Synthetic sequences and thiolated sequences (1&2) were purchased from IDT Korea. Gold nanoparticles (30 nm) were purchased from BBI Solutions.
3. *A. Procedure (adapted from reference 1)*
   1. 1. Lyophilize 5 nmol thiolated oligonucleotide probe (TS1: 3’-tgc cgg tca cgg ccg ctg tc-5’ and TS2: 3’-atc gtt ggg gtg acc gag cc-5’).
      1. a. Steps 1-20 were conducted for TS1 first and subsequently repeated for TS2.
   2. 2. Prepare 1 ml of 0.1 M DTT solution in the disulfide cleavage buffer.
      1. a. This solution must be made fresh every time.
   3. 3. Add 100 µl DTT solution (as prepared in Step 2) to the 5 nmol of lyophilized DNA, wrap in foil and let stand at room temperature for 2–3 h. Vortex occasionally.
   4. 4. 15 min before the completion of disulfide cleavage, begin flushing a Nap-5 column with NANOpure water. At least three column volumes of NANOpure water must flush through before adding DNA.
   5. 5. Add the 100 µl of DNA to the column after all the water has run through.
   6. 6. Once the 100 µl of DNA has flowed into the column, add 400 µl of NANOpure water to the column and allow it to flow through uncollected.
   7. 7. Then add 950 µl NANOpure water to the column, and collect the flowthrough 3–4 drops at a time in 1.5-ml microcentrifuge tubes.
   8. 8. Use a UV-visible spectrophotometer and the absorbance at 260 nm to determine the DNA location and concentration using Beer’s Law: A = εCι, where ε is molar absorptivity, C is concentration and ι is cell path length.
   9. 9. Rinse a 20-ml EPA vial with ethanol and then NANOpure water; blow dry.
   10. 10. Add 1 ml 13-nm Au-NPs synthesized previously.
   11. 11. Calculate the number of moles of oligonucleotide per tube, and add 4 nmol of the freshly reduced thiolated oligonucleotide to the Au-NPs. Record the volume.
   12. 12. Wrap in foil and place on an orbital shaker overnight at room temperature.
       1. a. PAUSE POINT: This step can be left overnight.
   13. 13. Add phosphate adjustment buffer to the NP solution to obtain a final phosphate concentration of 9 mM.
       1. a. Calculation: 1,000 µl AuNPs + x µl DNA = total volume in µl. (Total volume 1 in µl)/10 = y µl phosphate adjustment buffer needed.
   14. 14. Add surfactant solution to obtain a final SDS concentration of ~0.1% (wt/vol). This helps to keep the particles from aggregating and adds to the efficiency of washing them in future steps.
       1. a. Calculation: 1000 µl AuNPs + x µl DNA + y µl phosphate adjustment buffer = total volume 2. SDS to add = (total volume 2 × 0.1)/10.
   15. 15. Rewrap in foil and place on an orbital shaker for 30 min.
   16. 16. Calculate the volume of salting buffer needed to obtain a final concentration of 0.3 M NaCl.
       1. a. Calculation: (Total volume 2 × 0.3 M)/(2M) = volume of salting buffer needed in microliters. The number of additions of the salting buffer is equal to 6, therefore the amount per addition is equal to the volume of the salting buffer divided by 6.
   17. 17. Over the course of 2 d, make six additions of one-sixth of the total salting buffer needed to reach a final concentration of 0.3 M NaCl. Do the additions while shaking gently or at a low vortex speed.
   18. 18. After the last salt addition, allow the particles to equilibrate overnight. Well-functionalized Au-NPs should be the same color as the un-modified Au-NPs with no visible aggregates.
       1. a. PAUSE POINT: Particles can be stored at room temperature for as long as 1 month in this state.

TARGET DNA/FUNCTIONALIZED GOLD
NANOPARTICLES HYBRIDIZATION

1. *A. Equipment*
   1. Vortex machine, orbital shaker
2. *A. Materials*
   1. Synthetic sequence (SS): 5’-acggccagtgccggcgacagctctagcaaccccactggctcgg-3’, PBS (pH 8), 1.5mL microcentrifuge tubes, labelling tape, purified water

      Synthetic sequences and thiolated sequences (1&2) were purchased from IDT Korea.
3. *A. Procedure*
   1. 1. Mix in 1.5-ml microcentrifuge tubes: (i) 30 µl PBS (8) buffer; (ii) 10 µl GNPs solution; (iii) 10 µl target solution.
   2. 2. Shake the reactions at room temperature for 45 min. Shake sufficiently so that the particles do not settle.
   3. 3. Use a UV-Visible spectrometer to determine shifts in absorbance of samples.

REFERENCES

1. 1.
   [Hill, H., Mirkin, C. The bio-barcode assay for the detection of protein and nucleic acid targets using DTT-induced ligand exchange. Nat Protoc 1, 324–336 (2006).](https://doi.org/10.1038/nprot.2006.51)
2. 2.
   [Zagorovsky, K. and Chan, W.C.W. (2013), A Plasmonic DNAzyme Strategy for Point-of-Care Genetic Detection of Infectious Pathogens. Angew. Chem. Int. Ed., 52: 3168-3171.](https://doi.org/10.1002/anie.201208715)
3. 3.
   [Mereuta, L., Asandei, A., Dragomir, I.S. et al. Sequence-specific detection of single-stranded DNA with a gold nanoparticle-protein nanopore approach. Sci Rep 10, 11323 (2020).](https://doi.org/10.1038/s41598-020-68258-x)
4. 4.
   [Wang, M., Zhang, H., Zhang, W., Zhao, Y., Yasmeen, A., Zhou, L., Yu, X., & Tang, Z. (2014). In vitro selection of DNA-cleaving deoxyribozyme with site-specific thymidine excision activity. Nucleic acids research, 42(14), 9262–9269.](https://doi.org/10.1093/nar/gku592)

YONSEI\_KOREA 2021
![](https://static.igem.org/mediawiki/2021/b/b7/T--Yonsei_Korea--main_logo_1.png)

Yonsei\_Korea is a convergent team of students based in Seoul, South Korea striving to create meaningful change utilizing synthtic biology in the iGEM 2021 Competition.

* Experiments
* DNAzyme CLEAVAGE Cleavage Assay
* Agarose Gel Electrophoresis
* Page
* Denaturing Page
* DNA Functionalized
* Target DNA/Functionalized
* References

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Yonsei Korea/Results

IGEM\_YONSEI

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In the Laboratory:

The **Science**
Behind It All

RESULTS

OVERVIEW

    In this section, we plan to analyze the most important results that we obtained from our experiments. This is divided into two sections largely: part 1 is about DNAzyme cleavage assay results and part 2 is about nanoparticles. In part 1, we have analyzed PAGE and urea PAGE results. First, we have interpreted and analyzed each band on the gel. Second, to quantify the amount of DNA present in each band, we measured the net band intensity with Fiji, an open source program. In part 2, we have been expecting to yield results from our nanoparticles experiments; however, we are still in the process of them.

PART 1: DNAzyme CLEAVAGE ASSAY

Native PAGE results

    Samples DNAzyme-gene (substrate) A were reacted with 1x, 2x, and 5x reaction buffers. For each concentration of reaction buffer, 4 samples were tested: A-1, A-2, A-3, and A-4. Details about each sample can be found below:

| 1 | DNAzyme only |
| --- | --- |
| 2 | Gene (substrate) only |
| 3 | DNAzyme + gene, but without catalyzing the cleavage reaction |
| 4 | DNAzyme + gene, with catalyzing the cleavage reaction |

    After the cleavage reaction, it was visualized with native PAGE (12% and 15%). The gel was run at 90V for 120 minutes. The figure below represents the native PAGE results.

![igem_yonsei](https://static.igem.org/mediawiki/2021/8/86/T--Yonsei_Korea--results_1.png)

*1. Interpretation & analysis of bands*
    It was possible to observe bands at 4 distinct locations, which are (a), (b), (c), and (d) in Figure 1. (c) and (d) each represents DNAzyme A and Gene A. (a) is observed in A-3 and A-4, but not in A-1 and A-2. Therefore, (a) would represent DNAzyme-Gene A hybridized, but not reacted or cleaved. This is because DNAzyme requires specific ions such as Cu2+ and Mn2+ to exercise its cleavage activity. However, since none of these are present in A-3, DNAzyme would not be catalyzed, and therefore would not cleave the gene (substrate) even though the two stands are hybridized with each other. (b) is only observed in A-4, and this represents that the cleavage activity occurred. Since A-4 contains important ions such as Cu2+ and Mn2+, activated DNAzyme could cleave the gene (substrate). As gene (substrate) is cleaved at CTGC by DNAzyme, this 4bp would be released and the structure of the DNAzyme-gene complex would change. In other words, the cleaved gene except for CTGC is still hybridized with the DNAzyme binding arms, but has a different secondary structure compared to unreacted DNAzyme-gene hybridized structure. Since migration in PAGE is also affected by structure as well as size, the resulting product would be present at (b).

The diagram below represents possible structures that might have formed:

1. ● Green: DNAzyme
2. ● Yellow: Left-binding arm of the target gene
3. ● Red: CTGC sequence - sequence where DNAzyme specifically recognizes the target gene
4. ● Orange: Right-binding arm of target gene

![igem_yonsei](https://static.igem.org/mediawiki/2021/5/53/T--Yonsei_Korea--results_2.png)

*2. Quantification of the band intensity*
    Using Fiji, an image analysis software, we measured the light intensity of each of the bands (Figure 2a). Fiji measures the mean of the light intensity of the pixels within the selected area. The area of measurement was kept constant as 925. The result is shown as in Figure 2b (15% PAGE) and 2c (12% PAGE). (a), (b), (c), (d) represent the same thing as above.

The quantification process was conducted as:

1. 1) Since the background is blue, adjust the picture into an 8-bit image.
2. 2) Measure the mean light intensity of the background and each band.
3. 3) Subtract the background value from the value of each band to calculate the net band intensity.

![igem_yonsei](https://static.igem.org/mediawiki/2021/f/ff/T--Yonsei_Korea--results_3.png)
![igem_yonsei](https://static.igem.org/mediawiki/2021/7/7d/T--Yonsei_Korea--results_4.png)

*3. Analysis*
    With the band intensity measurement results, we compared and analyzed the following:

1. ● Comparison between A-1 and A-4 at each concentration of reaction buffer: this would allow us to see whether or not our designed DNAzyme participates in the cleavage reaction.
2. ● Comparison between A-3 and A-4 at each concentration of reaction buffer: this would allow us to see whether or not our designed DNAzyme exercised the cleavage activity.

![igem_yonsei](https://static.igem.org/mediawiki/2021/8/8b/T--Yonsei_Korea--results_15.png)

    As shown in Figure 4, the net band intensity of A-4 at (c) is decreased compared to the net band intensity of A-1 at (c). Since (c) represents solely the DNAzyme, this clearly shows DNAzyme participated in cleavage reaction. In 15% PAGE, the net band intensity of A-1 is almost the same; however, the net intensity of A-4 at (c) decreases more, as the reaction buffer concentration increases. This is because as the concentration of the reaction buffer increases, the amount of important ions (Cu2+ and Mn2+) increases, which means that more DNAzyme will be activated, since they require these ions for catalytic activity. However, this trend is not quite observable in 12% PAGE: the net band intensity of A-1 is low in 5x reaction buffer condition. As these samples were run at the end, it was hard to obtain a high quality picture of them, since the light intensity of the picture at the end is diminished compared to the other lanes.

![igem_yonsei](https://static.igem.org/mediawiki/2021/e/ec/T--Yonsei_Korea--results_6.png)
![igem_yonsei](https://static.igem.org/mediawiki/2021/e/e9/T--Yonsei_Korea--results_7.png)

    As shown in Figure 3, the net band intensity of A-4 at (b) is higher than the net band intensity of A-3 at (b). Since (b) represents the cleaved DNAzyme-gene (substrate), this clearly shows that DNAzyme exercised cleavage activity in the presence of the ions (Cu2+ and Mn2+) in the reaction buffer.

*4. Summary of the results*

1. ● It is clear that the DNAzyme participates in the cleavage reaction, and this amount is proportional to the concentration of the ions that catalyze its cleavage activity.
2. ● DNAzyme cleavage activity is present only in the presence of certain ions. Without it, DNAzyme and substrate only hybridize: no cleavage activity is observable.

Urea PAGE results

    Sample DNAzyme-gene (substrate) A was reacted with a 2x reaction buffer. Here, a total 7 samples were tested: A-1 to A-7. Unlike in native PAGE, only the 2x buffer was used. Details about each sample can be found below:

| 1 | DNAzyme only |
| --- | --- |
| 2 | Gene (substrate) only |
| 3 | DNAzyme + gene + 2x reaction buffer without CuCl2 *(DNAzyme : gene = 1 : 0.1)* |
| 4 | DNAzyme + gene + 2x reaction buffer with CuCl2 *(DNAzyme : gene = 1 : 0.1)* |
| 5 | DNAzyme + gene + 2x reaction buffer with CuCl2 *(DNAzyme : gene = 1 : 0.25)* |
| 6 | DNAzyme + gene + 2x reaction buffer with CuCl2 *(DNAzyme : gene = 1 : 0.5)* |
| 7 | DNAzyme + gene + 2x reaction buffer with CuCl2 *(DNAzyme : gene = 1 : 1)* |

    Samples A-4, 5, 6, 7 all contain 2x reaction buffers including CuCl2, but the ratio between DNAzyme: gene concentration varies. However, the concentration of DNAzyme is kept constant. A-4 has the minimum concentration and A-7 has the maximum concentration of gene A.

After the cleavage reaction, it was visualized with urea PAGE (8%). The gel was run at 200V for 40 minutes. The figure below represents the urea PAGE results.

![igem_yonsei](https://static.igem.org/mediawiki/2021/4/4c/T--Yonsei_Korea--results_8.png)

*1. Interpretation & analysis of bands*
    Neglecting side bands and noises, it was possible to observe meaningful bands at 4 distinct locations, which are (a), (b), (c), and (d) in Figure 5.
    Some things to note is that although urea PAGE was used to separate possible hybridization complexes into single stranded DNA, the results showed that denaturing did not occur properly. In other words, the urea within the gel mesh would not be useful because the denaturation was not complete during the sample preparation step. To get rid of this, we could extend the denaturation time for 30 minutes or more. Also, since our target gene substrate and DNAzyme is single-stranded as its own, it could have formed additional secondary structures on its own. This led us to observe a similar band pattern as in native PAGE results.

    In addition, a lot of side bands and noises were detected, as seen at the upper and lower part of the urea PAGE gel. We ordered our DNA products without HPLC purification, meaning that other truncated synthesis products were not removed. Ensuring the high purity of our samples was not possible due to funding constraints, so this could have affected our results. This was evident from the appearance of several unspecific bands (contributing to a high background noise) in all lanes including the control groups: ssDNAzyme and single-stranded target gene.

    (c) and (d) each represent DNAzyme A and Gene A. (a) is observed in A-3 and A-4, but not in A-1 and A-2. Therefore, (a) would represent DNAzyme-Gene A hybridized, but not reacted or cleaved. This is due to the same reasons as in the native PAGE section. However, (b) appeared in urea PAGE, which was not present in native PAGE results. Unlike the thin band (b) in native PAGE, 2 bands were overlapping in urea PAGE (b). It is difficult to observe the 2 overlapping bands when the gene (substrate) concentration is low, but it becomes clear as the concentration increases, as shown in A-7 sample. (b) represents the cleaved products: the intensity is very weak in A-3, whereas it is clearly visible in A-4 to A-7. In fact, the weak intensity of (b) at A-3 might be just noise, as the intensity is very dim. The cleaved products (b) in urea PAGE would be analogous to (b) in native PAGE, although there is a difference in the appearance and the location of the bands. Although denaturation by urea was not 100% efficient, urea definitely would have affected the structure of the cleaved DNAzyme-gene structure, resulting in a different structure from (b) in native PAGE.

The diagram below represents possible structures that might have formed:

1. ● Green: DNAzyme
2. ● Yellow: Left-binding arm of the target gene
3. ● Red: CTGC sequence - sequence where DNAzyme specifically recognizes the target gene
4. ● Orange: Right-binding arm of target gene

![igem_yonsei](https://static.igem.org/mediawiki/2021/a/ad/T--Yonsei_Korea--results_9.png)

*2. Quantification of the band intensity*
    As we did in native PAGE result analysis, we used Fiji to measure the light intensity of each of the bands (Figure 6a). The area of measurement was kept constant as 940. The result is shown in Figure 6b. (a), (b), (c), (d) represent the same thing as above.

The quantification process was conducted as:

1. 1) Measure the mean light intensity of the background and each band.
2. 2) Subtract the background value from the value of each band to calculate the net band intensity.

![igem_yonsei](https://static.igem.org/mediawiki/2021/0/0c/T--Yonsei_Korea--results_10.png)
![igem_yonsei](https://static.igem.org/mediawiki/2021/d/d5/T--Yonsei_Korea--results_11.png)

*3. Analysis*
    With the band intensity measurement results, we compared and analyzed the following:

1. Comparing (b) net band intensities for A-3 to A-7 to check the following:
2. ● Whether or not our DNAzyme is specifically catalyzed by Cu2+
3. ● Whether or not DNAzyme cleavage activity of proportional to the amount of DNA (substrate) present

![igem_yonsei](https://static.igem.org/mediawiki/2021/8/87/T--Yonsei_Korea--results_12.png)

*Cu2+ requirement for DNAzyme catalytic activity*
The difference between A-3 and A-4~A-7 was the presence of Cu2+ ions in the reaction buffer: in A-3, all the other ions such as Mg2+, Na+, K+, Mn2+ were present, except for Cu2+. When we compare the net band intensity of A-3 to others (A-4~A-7), the net band intensity of A-3 is significantly low. However, the catalytic activity of A-3 is not exactly zero: due to Mn2+, it was possible for the DNAzyme to cleave the gene (substrate), but with a very low efficiency. Therefore, in order for our DNAzyme to be efficient in cleaving DNA, Cu2+ is essential.

*DNAzyme cleavage activity is proportional to the amount of DNA (substrate)*
Samples from A-4 to A-7 had an increasing concentration of the target substrate, while that of DNAzyme remained unchanged (10μM). The 1:1 ratio between DNAzyme and substrate (10μM:10μM) was the most effective reaction since we observed a shrunken band size at (b) (represents DNAzyme) compared to other samples. This indicated that DNAzyme cleaved the available substrate fully, leaving only unreacted DNAzymes. Therefore, it is possible to conclude that our detection system based on the DNAzyme cleavage assay is effective and efficient.

*4. Summary of the results*

1. ● Cu2+ is specifically required for the DNAzyme cleavage activity, since it highly increases the efficiency of cleavage. Without Cu2+, cleavage activity is hardly observable.
2. ● DNAzyme cleavage activity is proportional to the amount of DNA (substrate present), which makes the detection system based on the DNAzyme cleavage assay effective and efficient.

PART 2: NANOPARTICLES FUNCTIONALIZATION

UV-Vis absorption sample DNA quantification

    Phase 1 of using gold nanoparticles for our detection system is to functionalize them with our 20 base pair complementary sequences to our target gene. Here we began the salt aging method to functionalize both thiolated DNA sequences TS1: 3’-tgccggtcacggccgctgtc-5’ and TS2: 3’-atcgttggggtgaccgagcc-5’ to two samples of gold nanoparticles. In total we have four samples (2 for TS1 and 1 for TS2). This enables better chances of proceeding with better functionalized gold nanoparticles as some samples could aggregate early on in the experiments.

1. 1. Sample A1: 3’-tgccggtcacggccgctgtc-5’ functionalized
2. 2. Sample A2: 3’-atcgttggggtgaccgagcc-5’ functonalized
3. 3. Sample A3: 3’-tgccggtcacggccgctgtc-5’ functonalized
4. 4. Sample A4: 3’-atcgttggggtgaccgagcc-5’ functonalized

    To functionalize our gold nanoparticles, first the oligonucleotide probes were prepared with PBS buffer (pH 8) and purified water. In order to functionalize them to our GNPs we needed to determine which samples had the DNA present in them; therefore, we used a nanodrop UV-Vis machine to detect the DNA absorbance at 260nm. The results were obtained as followed for all of our samples:

![igem_yonsei](https://static.igem.org/mediawiki/2021/2/26/T--Yonsei_Korea--results_13.png)

    Some samples did not contain DNA while others contained it, so we employed those with the DNA further in the experiment.

    In the next steps, phosphate adjustment buffer (final 9mM), surfactant solution (final ~0.1% (wt/v)) and salting buffer (final 0.3M NaCl) were added with the DNA and gold nanoparticles. The salting buffer was added six times over the course of two days. Our gold nanoparticles did not have any visible aggregates except for one sample, meaning that they have been properly functionalized.

![igem_yonsei](https://static.igem.org/mediawiki/2021/4/49/T--Yonsei_Korea--results_14.png)

    Due to the wiki freeze deadline, we are still in the process of the next phase to hybradize our functionalized gold nanoparticles with our target gene and observe a color change from red to purple. We shall present these results in time for the Giant Jamboree.

FUTURE PLANS

    Our DNAzyme needs to be checked against another substrate sequence, to confirm that exact sequence specificity is required for cleavage activity. Moreover, our gold nanoparticles should be further hybridized with our target gene to induce a color change from red to purple.

CONSIDERATIONS FOR REPLICATING THE EXPERIMENTS

    In conducting the DNAzyme cleavage assay, it is important that all DNA samples are kept on ice while performing the experiments. Otherwise, it would cause DNA degradation, leading to inexact results. In addition, the denaturing step in urea PAGE should be done under a sufficient time. Since DNAzyme and target gene DNA are single stranded, there is a higher possibility of forming secondary structure on its own.

YONSEI\_KOREA 2021
![](https://static.igem.org/mediawiki/2021/b/b7/T--Yonsei_Korea--main_logo_1.png)

Yonsei\_Korea is a convergent team of students based in Seoul, South Korea striving to create meaningful change utilizing synthtic biology in the iGEM 2021 Competition.

* Results
* Overview
* DNAzyme Cleavage Assay
* Native Page Sesults
* Urea Page results
* NanoParticles Functionalization
* Future Plans
* Conclusion