Difference between revisions of "Team:Yonsei Korea/Engineering"

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:

PHASE ONE        

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:

    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.

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 the 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:

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:

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

4. Summary of the 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:

    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.

1. nterpretation & 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:

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:

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

4. Summary of the results

PART 2: NANOpARTICLES FUNCTIONALIZATION        

    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.

    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:

    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.

    Currently we are continuing 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.