Team:NTHU Taiwan/Human Practices
Meet up with The Society of Wildness
Date：2021.08.10
For most projects which aim to tackle environmental issues, the most important of all for the conductors is to know the opinion of the public. Therefore, we met the chairman of The Society of Wildness, which is one of the most famous environmental conservation groups of our countries composed of people who are concerned about environmental issues.
She told us that the top priority of all is to make sure not to hurt the forest itself. Upon hearing about this, we decided not to conduct our project on the pine tree first; instead, we chose the pine callus, which we expected can simulate the protection of the pine tree against B. xylophilus without the concern about hurting the environment.
Meet up with Our Island
Date：2021.08.18
We also met the reporters and producer of Our Island, a TV show of our country which also concern a lot about the environment.
They told us that in order to make the public more curious about an issue, we should make our description easier the be understood； therefore, one of our model designs, which is mainly to realize the efficiency of RNAi after being uptaken by B. xylophilus, simplify body of it into several regions and visualize the process in order to let it better understood by the general public.
A questionnaire about PWD
Date：2021.09.29
As an environmental issue, we should know the viewpoint and prior knowledge of the public about it; therefore, to realize their recognition about the Pine Wilt Disease(PWD), we conducted a questionnaire survey about it, and received around 300 effective samples. Next, we analyzed the difference between their response toward the importance of pine wilt disease and methods to deal with it.
Since the public is composed of various groups of experts in various fields, we wrote a brief article to simply explain what PWD is and the various solutions to deal with it. We wondered about the importance of PWD in the public’s viewpoint, so both before and after reading the article, we asked them how important PWD was in their mind, 1 for least important, 5 for very important. As the result shown on the left, we can see that the average grade of the importance of PWD has risen after reading our article.
We also conducted a one-sample t-test on the average of both groups; the null hypothesis is that the average of both groups are the same. As the result on the right, the p-value of the t-test is much smaller than 0.05, so we rejected the null hypothesis. To conclude, people have raised their awareness toward PWD through the process of reading the article.
As for the idea about the ways to deal with the pine wilt disease, we listed three common solutions: physical method, chemical method and biological method, to know people’s understanding about these three methods. Commonly, people were not familiar with certain methods before reading our article, which explains it in detail. The biological method was least understood by the public; after all, it is not easy to remember a complicated technique just through normal news or magazines. However, after knowing that the existing methods can’t solve the problem fundamentally by our brief article, the public turned to the biological method. From the pie chart below, we can know that the public wanted to see an environmentally friendly and efficient solution to PWD.
We also conducted a one-sample t-test to compare their idea for the new method, the null hypothesis is three of the methods have the same amount of response. (286/3=95.3333)
Here is the result: the p-value is very small, so we rejected the null hypothesis. The biological method was obviously higher than the average of the other methods.
Integrated Human Practice
Project content and feasibility
Dr. Ya-Tang Yang
Professor from Department of Electrical Engineering
National Tsing Hua Univerisity
Prof. Yang’s lab focuses on Microfluidic and BioMEMS, including an integrated electrokinetic droplet generation system, novel electronics-based bioreactor, and synthetic biology for carbon fixation.
As soon as we come up with the simple contour of our project, we come to meet our PI, who is an expert in Microbial Electromechanical, a field that combines biology and engineering. He reminds us of the cost of time to get one set of data, what’s more, he also gives us some advice about our dry lab about the detection of the wound for pine trees.
Takeaway:
- Change in software design
Because of his help, we found some research from another school that also discusses a similar problem. Most importantly, for the detection part, we found that it costs so much time and human practice to use AI train for the analysis； therefore, we did not take the method at last.
Dr. Yu-Chun Lin
Associate Professor of Institution of Molecular Medicine
National Tsing Hua Univerisity
Professor Lin’s lab mainly studies cell biology, molecular biology, cell imaging technology, biotechnology, and bioengineering. Also, they focus on the research about Remotely controlling of cells.
Prof. Yu-Chun Lin was once the PI of another iGEM team and majored in synthetic biology. He told us to find the core part of our project to verify for better persuasion. And the simplicity of the experiment is also important in order to quantify the amount of RNA uptake. As an expert who gallops in the field of biosynthesis, he also reminds us to notice the difference in the construction of a plasmid that needs to be transferred to plant callus through Agrobacterium tumefaciens-mediated plant transformation, such as the selection of promoter.
Takeaway:
- Better expression method for project result presentation
- More feasible construction idea
Thanks to his advice, we make the presentation of our result more simple by using the comparison method between the modified group and the control group.
During the construction, we choose the promoter which induces the encode of our sequence in the plant. Also, we choose the plasmid which contains T-DNA, the indispensable component upon using Agrobacterium to transform the plasmid.
Dr. Chung-Yu Lan
Professor Institute of Molecular and Cellular Biology
National Tsing Hua University
Prof. Lan’s lab is studying different aspects of microbial pathogens and pathogen-host interactions. The current focus of research includes antimicrobial peptides, biofilm/drug resistance, and signaling/regulation of virulence gene expression. They take a multidisciplinary approach in studies including molecular biology, genetics, biochemistry as well as functional genomics.
Prof. Lan, who is an expert in C. albicans, reminded us of some questions related to plasmid construction and the target concentration for RNAi. First of all, he told us to emphasize the importance of our project. And asked us to clarify the relationship between nematode, pine, and our design, which means how to reach enough concentration to avoid the spreading of B. xylophilus. Last but not least, there may have several genes which have similar functions to our target genes and it may be difficult if we construct all our target genes in a plasmid.
Takeaway:
- Construction adjustment and model design
Due to his suggestion, we decided to construct a plasmid with the target gene separately in order to get a better result. We also construct a model for realizing what concentration of RNAi should we add in an attempt to kill the nematodes.
Plant Biology
Dr. Tzu-Yin Liu
Associate Professor of plant molecular and cell biology
National Tsing Hua University
Professor Liu is a specialist in plant biology and plant biotechnology, her research is focused on the molecular mechanism of plant response to surrounding stress, using Arabidopsis as a model organism.
Plant synthetic biology is a sophisticated field of research, the generation of the plant is much longer than bacteria and the culture condition must be under precise control. Prof. Liu shares her lab experience in the transformation and regeneration of Arabidopsis and emphasizes the physiological differences between herbaceous plants and woody plants, especially the concern to Agrobacterium-mediated transformation. She also suggests that the feasibility of our project depends on how pine trees will respond to our engineered parts that have been integrated into the plant genome. Like other gene-modified crops, the plant might face a tradeoff between the advantages of the new trait and meanwhile the energetic cause of continuing expressing foreign genes, the dsRNA coded nematode protein in our case.
Takeaway:
- Revise of Agrobacterium transformation protocol
- Streamlining process
Dr. Liu gives us several pieces of advice on the experiment design, helping us to improve the project.
One of the biggest challenges in our project is to put the binary vector into Agrobacterium competent cells. Initially, we plan to use triparental mating that involves a donor strain, a helper strain, and a recipient strain. Dr. Liu indicates that there are other transformation methods which are more simplified and efficient, such as the freeze-thaw method or electroporation. These systems are widely used in Agrobacterium, providing us a more reliable way to conduct the experiment.
At the early stage of our project discussion, we considered inoculating on pine tree sapling to verify the effect of nematode RNA interference. After the interview, we think that the limitation of competition time is a serious issue concerning plant generation. Dr. Liu suggests that the antibiotics selection followed by plant regeneration is extremely time consuming. Therefore, focusing on the callus culture stage is much more practical to a short-term iGEM project. Assays like GUS staining or qPCR might be a possible option for us as a proof of project concept.
Prior Knowledge to Nematode (Caenorhabditis elegans)
Dr. Oliver Wagner
Professor of Molecular Cell Biology, National
National Tsing Hua University
Professor Wagner’s lab is using the model organism C. elegans to study the nervous system and the regulation of motors involved in various neurodegenerative diseases.
Nematode C. elegans is a common model organism frequently used in biological research, it is also the original organism on which scientists thoroughly study RNA interference mechanisms. Thus, consulting with professors specialized in C. elegans allows us to have a general idea about how these tiny animals help the study of genetics at the molecular level.
The interview with Dr. Wagner was like a small rehearsal for the iGEM presentation, the professor invited his lab members to come together. That was the first time we tried to deliver the whole project in English and get feedback from student audiences other than a lab PI. Moreover, Dr. Wagner was in earnest to provide us technical support after listening to our presentation. He offered us an opportunity to visit the lab to learn nematode cultivation techniques.
Takeaway:
- Learning nematode cultivation
- Reorganizing our project presentation
Dr. Wagner kindly provided us professional help that equipped us with the necessary skill for nematode experiments.
The major concern for our project is the organisms we might use in addition to E. coli during the experiment. However, Dr. Wagner’s lab shed a light on us, making us decide to continue on this project with nematodes involved. Our wet-lab member visited Wagner’s fellow and learned the subculture technique of C. elegans in person. They also gave us some suggestions of potential analytic assays we might need to verify our result, such as the western blot and qPCR of nematodes.
As described above, comments from students in Wagner’s lab were very helpful for us to improve. For example, some molecular mechanisms in plants might need a more detailed explanation for a general audience from different backgrounds, the introduction of the main issue must be precise to the point, not to mention our English speaking skill that is in urgent need to be improved.
Dr. Horng-Dar Wang
Professor and Department Chair of Life Science
National Tsing Hua University
Professor Wang and his fellow researchers are dedicated to understanding the underlying molecular mechanism for regulation of longevity and anti-aging drug development, using Drosophila and C. elegans as model organisms.
Our discussion with Dr. Wang was focused on the details of a possible dsRNA soaking experiment, which is used to knockdown target genes in nematodes. He points out a few important aspects we have to affirm before the experiment. For instance, the optimized promoter for dsRNA in vitro synthesis, the concentration of dsRNA for soaking, and the degradation time of dsRNA either in vitro or in vivo. In addition, Dr. Wang also suggests that data from the soaking assay could be applied as a control to infer the actual uptake rate in the future experiment. The interview with Dr. Wang makes us organize our thoughts carefully and take a step toward a feasible iGEM project.
Takeaway:
- A way toward B. xylophilus
Dr. Wagner kindly provided us professional help that equipped us with the necessary skill for nematode experiments.
Although C. elegans is a general and powerful tool to study RNAi mechanisms, it still has many genetic and physiological differences compared with B. xylophilus, our target plant-parasitic nematode. These divergences of nematode species are a particularly significant problem in our project since the 20nt base pair of dsRNA has high specificity toward the B. xylophilus genome. Thus, Dr. Wang kindly contacted the Taiwan C. elegans community, asking if there is anyone who also studies B. xylophilus in the lab. Fortunately enough, we got the contact information of Dr. Yang from National Taiwan University, who works on parasitic nematodes, including B. xylophilus.
The Pine Wilt Nematode - Bursaphelenchus xylophilus
Dr. Jiue-In Yang
Associate Professor of nematology
National Taiwan University
The Nematology and Microbial Ecology Laboratory is dedicated to studying the interaction between parasitic nematodes, plants, and microbes. Yang's research also includes the pathogenic mechanism of nematodes and potential nematode management strategies. One of the parasitic nematodes they are interested in is the B. xylophilus which causes pine wilt disease.
Consulting with Dr. Yang and her graduate student helped us to clarify the concept about the whole mechanism of pine wilt disease, how nematodes invade pine trees, what attempts trees make to fight against the uninvited guest, and why they would die eventually. Besides, she also provides us a brief review of the pine wilt disease research in Taiwan, covering the species-specific infection of the host plant, diagnostic methods, and potential treatments. There are also several things we need to consider when we build up our models, such as the difference of vector beetles between countries, seasonal factors in the migration of beetles , and the nematodes life cycle.
Takeaway:
- Obtain the pine wilt nematode
- The Alternaria citri cultivation
- The B. xylophilus cultivation
- RT-qPCR protocol
We got lots of practical help from Dr. Yang that made our project more feasible.
Unlike C. elegans, B. xylophilus is a parasitic plant nematode that causes disease, so we must pay attention to safety during the experiment. We must obtain the nematode sample from a reliable laboratory, and the lab work should be under supervision.
The pine wilt nematodes cultivated in labs are usually fed on fungi instead of plants. Dr. Yang gave us an A. citri pure culture tube for nematode cultivation. They also kindly record a video demonstrating the detailed subculture process step by step. Thereby we could build up a culture system in our iGEM lab.
As long as we have well-prepared A. citri slant tubes, we can inoculate the nematodes. The nematode subculture also requires proper aseptic technique, taught by students from Yang’s lab.
In our project, we want to measure the changes in gene expression of nematodes after they uptake dsRNA from the engineered callus. Dr. Yang provided us with their qPCR protocol which contains optimized reaction conditions for B. xylophilus.