Biosafety

New Parts and Organisms Introduced

We are aware that with everything we handle in the lab there are certain risks to be evaluated. Here we carefully lay out the main features and origins of the new parts in the project and the purposes of use. Our main chassis throughout our project, including the lab training session, was Escherichia coli DH5alpha commercial competent cells. We were fully aware that E. coli may cause irritation to skin, eyes, and respiratory tract, and may also affect kidneys; therefore we wore proper personal protective equipment while conducting experiments.

*Note: In this project we used only Risk group 1 organisms (Risk group source: NIH). The spreadsheet attached is the one we filled for the final safety form. NYMU-Taipei Safety2015 Spreadsheet

Prevention

In the prevention stage, we extracted FYVE from the Mus musculus Hrs cDNA to engineer it into an artificial competitive inhibitor against the Phytophthora infestans effector protein. Hrs (Hepatocyte growth factor-regulated tyrosine kinase substrate) is a key component of the endosomal sorting complexes required for transport and regulates endocytosis/exocytosis and the accumulation of internal vesicles in multivesicular bodies.[1] We acquired the cDNA from Professor Ming-Ji Fan (National Yang Ming University Department of Life Sciences and Institute of Genome Sciences). Naturally, proteins containing the FYVE zinc finger domain have biological functions such as endocytic transport, regulation of endosomal membrane fusion, or signal transduction. Mice, the origin of the gene, however, may transmit diseases (E.g. lymphocytic choriomeningitis, rickettsialpox, leptospirosis, etc) and may also be a source of certain allergens.

After circuit construction, since our competitive inhibitor, the engineered dimeric FYVE, is of animal origin, we would like to test the function of it in plant cells. We transfected our construct into BY-2 protoplasts of tobacco (Nicotiana tabacum) to see if it worked in plant cells. We understood that all parts of tobacco contain nicotine, which may lead to addiction. To carry out this experiment, our team members contacted researchers in Academia Sinica in advance and was then granted to work in their laboratory.

Detection

To detect the infection of P. infestans, we first constructed our circuit in E. coli DH5alhpa, transferred it onto pBBR1, and then transformed the plasmid into E. coli S17-1, which was acquired from the laboratory in Academia Sinica and served as our chassis to carry out the conjugation experiment. We conjugate E. coli S17-1 with Shewanella oneidensis MR-1 strain JG700, which had mtrB knocked out from its genomic DNA, to build the soil-based microbial fuel cell (SMFC). We then extracted mtrB gene, originally an extracellular iron oxide respiratory system outer membrane component gene, from S. oneidensis MR-1 to reconstruct the mtr pathway in order to create oscillating electric currents. S. oneidensis MR-1 (JG700) was acquired from Professor Jeffrey A. Gralnick (University of Minnesota BioTechnology Institute) while S. oneidensis MR-1 was acquired from the Bioresource Collection and Research Center. The Gram-negative bacteria S. oneidensis does not pose much threat to human health.

Cure

Defensin (Lm-def) was an antifungal peptide isolated from maca (Lepidium meyenii) and was used to treat the infection both in vitro and in vivo; however, in our project, the part was IDT-synthesized.

In order to test the antifungal activity of defensin, we acquired P. infestans from Taiwan Agricultural Research Institute. Although P. infestans is an aggressive plant pathogen, it basically does not do harm to human health; yet we have to be careful with containing the sporangia of P. infestans while doing related experiments. To test if our product functions, we inoculate P. infestans onto potatoes (Solanum tuberosum) strains Kennebec, Atlantic, and Tainong No. 1, with the former two acquired from Taiwan Agricultural Research Institute and the other from Dounan Town Farmers' Association.

We did learn that glucosinolates in maca may cause goiters when high consumption is combined with a diet low in iodine and that potatoes contain toxic compounds known as glycoalkaloids, of which the most prevalent are solanine and chaconine. Nevertheless, those factors only had minor effect on our experiment.

Our Chassis

1. E. coli strains: DH5alpha, S17-1, BL21 (DE3)
2. S. oneidensis MR-1 strain JG700
3. N. tabacum BY-2 protoplasts

We are genetically modifying E. coli DH5alpha for creating the oscillator system to test the optimum level of salicylic acid to make Nahr function best and also to express the engineered dimeric FYVE, which is used to prevent the binding of P. infestans effector protein and the potato PI3P phospholipid. By-2 protoplasts were modified via transfection to see if the gene of animal origin works well in plant cells. Other than that, we modify E. coli BL21 (DE3) to express the His-tagged protein, Lm-def, extracted from the plant maca (Lepidium meyenii).

Experiments on Organisms Other Than Our Chassis

Our bacteria are meant to carry the gene and transfer it into the potato cell; to see if the gene will express within plant cells, we test them in tobacco (Nicotiana tabacum) BY-2 protoplasts, as tobacco and potato both belong to the Solanaceae family and are biologically similar. For the transfection experiment, we have contacted research labs in Academia Sinica and are fortunately offered working area for the experiment. Apart from that, we will inoculate potato tissues with P. infestans to test if our product works in an isolated working place.

How Our Project Works

Our project consists of three major parts: prevention, detection, and cure. In the first stage, the bacteria will be used to carry the gene of interest, the FYVE coding sequence, into the potato cell via PEGylation. The FYVE domain binds to the potato PI3P phospholipid with higher affinity than the P. infestans effector protein RXLR domain, and can thus arrest pathogen infection. The second stage mainly features a soil-based microbial fuel cell. We utilize the mtrB gene extracted from S. oneidensis MR-1 to create an oscillator generating electric currents upon detection of salicylic acid. The microbial fuel cell then sends the signal to the producers to inform them of the infection. The last part, cure, is to treat the infected plant with defensin, an antifungal peptide originally existing in maca, and may serve as a more eco-friendly strategy to fight against potato late blight.

Reduction on the Risks Encountered

Handling risk group 1 organisms, we have all received safety training and always wear proper personal protective equipment. We carefully handle everything that may involve contact with our chassis organisms, inclusive of waste management, autoclave sterilization, and avoidance of contamination. Apart from that, as we would test the effectiveness of our product on P. infestans, we have found an isolated working place for conducting the experiments with P. infestans. We highly value biosafety problems as P. infestans pose great threat to plants. Since the P. infestans sporangia has a diameter of about 12~23 micrometers and the zoospores of about 15 micrometers, the built-in HEPA filter in our laminar flow hood, which, by US government standards, must be able to remove 99.97% of particles in the air passing through it with a size of 0.3 micrometer, should stop the pathogen to spread and cause harm to our natural environment.

Real Life Applications, Future Outlook and Risk Evaluation

1. In agriculture / on a farm
2. In the natural environment

Our system is built to battle against one of the world's most notorious plant diseases, the potato late blight. We try to solve the problem from several aspects, and fervently hope that our project can help farmers, manufacturers, and food processors handle this problem.

Our first stage of experiment, prevention, mainly features the creation of an engineered dimeric FYVE, which is used to compete against the binding of the pathogenic effector protein to the host PI3P phospholipid. However, in our future outlook, the development of transgenic potatoes requires careful evaluation, and we have contacted experts as well as anti-GMO non-profit organizations, and we would try to reduce its negative impact on other wild strains and the general environment. In the future we will keep on consulting Food Drug Administration and the Council of Agriculture in Taiwan and will continue advocating the legislation on related issues. For environmental risk assessment, we would follow the standards provided by WHO, which includes evaluation of the characteristics of the GMO and its effect and stability in the environment along with several other environmental factors to understand about the toxicity, allergenicity, and gene transfer and outcrossing of the inserted gene. As for the second stage, detection, the soil-based microbial fuel cells we created will be put on potato farms and the chassis organism, S. oneidensis MR-1 strain JG700, will be inoculated on the anode and be kept in a device that allows the exchange of substances but stops the bacteria from escaping. Lastly, when we cure the infected plant, we treat the plant with purified defensin. Defensin does have antifungal activities, but it can be degraded in the environment, and we only spray out defensin upon detection of the invading pathogen.

We hope to further explore the field of synthetic biology and combine various methods in different infection stages of the pathogen and to make greater use of our defense system to guard plants from diseases and to contribute more to global food security.