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From 2012.igem.org

Contents 1 Our iGEM Project: 2 Responses to Safety Questions 2.1 1. Would any of your project ideas raise safety issues in terms of: 2.2 2. Do any of the new BioBrick parts (or devices) that you made this year raise safety issues? If yes, 2.3 3. Is there a local biosafety group, committee, or review board at your institution? 2.4 4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering? Our iGEM Project: * Nitrogen Metabolism With an eye to combat the environmental issues related to nitrogen oxides, we have designed circuits that can produce nitrogen oxides reductases and transcriptional regulators into our organisms so that harmful nitrogen oxides can be reduced to nitrogen. Combined with the sulfur metabolism pathway and Calvin cycle that are inherent in cyanobacteria, we provide an eco-friendly, multi-function solution to the air pollution problem. This is a promising project with huge commercialized potential since the mass production of bioreactors full of our organisms is foreseeable. What is more interesting, with the help of division inhibitor, gene for invasion, we can install our designation into human cells as artificial organelles and grant human being the ability to survive in extreme environments such as Venus without wearing bulky space suits. * Sulfide Metabolism We all know that sulfide dioxides are one of the pollutants in urban area globally. In addition, sulfide compounds also exist on Venus. Therefore, we first think about how to reduce the sulfide dioxides in the atmosphere on earth; then we can move our project further on Venus. Our plan is to develop a system which can reduce the SO2 into H2S using the genes in microorganisms like sulfate reducing bacteria, such as Desulfovibrio desulfuricans. After we acquire H2S, we may use bacteria such as Oscillatoria limnetica, Rhodobacter capsulatus, and Cyanobacteria PCC7002 which contain Sulfide-quinone reductase(SQR).By using SQR, we can then use H2S as the reducing energy and can get carbohydrates such as glucose, and can provide them as the energy for creatures to survive. * Symbiosis Every year, iGEMers create plenty of bioparts. They code for peptides, proteins or composite parts for all kinds of functions. However, they are just DNA sequences. But what if we create parts at the scale of organelles? With this approach, we can implant any cells into the host depending on what kinds of functions we hope to see in it. Comparing with transforming cells using merely simple DNA sequence, implanting new organelles can bring more complicated functions and more precise controlling systems. We can dream about making eukaryote to fix nitrogen, reduced sulfite, or even making animals to photosynthesize! Or we can create organisms that can live in other planets!

Responses to Safety Questions

1. Would any of your project ideas raise safety issues in terms of:

• researcher safety,
• public safety, or
• environmental safety

Our projects are mainly associated with pollutent reduction and endosymbiosis. For discussing the safety issues connected with our project, this assessment will start by listing the organisms and biobricks we use, and develop in compliance with the guideline of iGEM Safety page correspondingly. The following organisms are species which we derived our biobricks from or practiced transformation and cloning. This list includes Desulfovibrio desulfuricans, Pseudomonas aeruginosa PAO1, Dictyostelium discoideum, Escherichia coli, Synechocystis sp. PCC 6803, Synechococcus elongatus PCC7942 and Synechococcus sp. PCC7002. None of the listed organisms is pathogens. On top of these new biobricks, we used some previous parts. These parts from iGEM kits are considerably safe.

Since our project is related to contaminant water, exhaust air and heavy metal, we need to thoroughly examine every process of the experiments we conduct. Under the instructions of our tutor, all transmissions and operations of biological materials meet the relevant laws and regulations. As a result of the heartily suggestion and assistance of our laboratory fellows, we developed a series of experiments to demonstrate our project without violating any national regulations or university requirements. The details of our experiments are available on the Experiment page.

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2. Do any of the new BioBrick parts (or devices) that you made this year raise safety issues? If yes,

• Did you document these issues in the Registry?
• How did you manage to handle the safety issue?
• How could other teams learn from your experience?

There is no any new BioBricks we made raise safety issue. They are different reductases from organisms mentioned above. Nitrate, nitrite, nitrous oxides reductase and derived from Pseudomonas aeruginosa PAO1. Thanks to Prof. Hwan-You Chang from National Tsing Hua University, we operated the transferring and cloning on purified genomic DNA. Therefore, we didn’t need to cultivate P. aeruginosa PAO1. The biobricks related to nitrogen reductase are ultimately transfected into the genome of Synechococcus sp. PCC7942, and all of the according procedures followed the strict regulations of BSL 1 (Basic Biosafety Level 1). The same operation standard (BSL 1) is also applied to experiments of the sulfate reducing enzymes derived from P. aeruginosa PAO1 as well. In addition, the sqr (sulfide quinone reductase) from Synechococcus sp. 7002, and like other biobricks mentioned above, it is expressed in Synechococcus PCC7942 following the same requirements too.

The other new biobricks we made includes zinTp, the promoter whose activity is induce by cadmium ion; and mnt protein, the ion transporter that pumps cadmium ions inside the bacteria. These genes to no harm to neither human being nor the environment.

Since there is little safety concern according to the properties of our new biobricks, we deduced that the new biobricks we submitted is safe and harmless.

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3. Is there a local biosafety group, committee, or review board at your institution?

• If yes, what does your local biosafety group think about your project?
• If no, which specific biosafety rules or guidelines do you have to consider in your country?

Center of Environmental Protection and Safety and Health is in charge of promoting and managing the safety and health issue in National Yang-Ming University. The following links will direct you to the website which contains regulations of biological safety issues. [http://ces-e.web.ym.edu.tw/front/bin/ptlist.phtml?Category=5 Center of Environmental protection and safety and health - Biological safety]. All things we do or conduct in our project should follow the rules.

Though most of our projects are considerably safe, one part of our projects involves in the manipulation of cadmium ions, which is restricted under the local regulations. With the help and advice of Center of Environmental Protection and Safety and Health, we conduct our cadmium related experiments in accordance with the associated rules.

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4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?

There is a part of our project specially dealing with biosafety issues to the environment. The Soil Cleaner, our project about collecting Cadmium, contains several interesting design to avoid gene flow and limit the leakage of artificial organisms into environment. We took advantage of previous biobricks to make sure the engineered E. coli can and can only live inside of Dictyostelium discoideum, which avoids unwanted gene exchange between our engineered E.coli and other wild type bacteria.

By cloning LLO and invasin into the E.coli, the bacteria can get into the amoeba and live inside it. Invasin helps the cell to be endocytosed by the host. Then Listeriolysin O makes the cell escaped from phagosomes in the cytoplasm of the host.

In case that the E.coli get rid of amoeba, we design a mechanism to cause the death of bacteria. Endolysin is an enzyme that degrades the bacterial peptidoglycan cell wall, resulting in lysis of the bacterial cell. Holins are small membrane proteins that let endolysin get to where it works. Anti-holin acts as anti-toxin by binding to holin, inhibiting it activity. LuxR promoter is a promoter which will only express downstream gene when the protein LuxR and signal molecule AHL, which is produced by protein LuxI, are both exist inside the bacteria. Engineering continual expression endolysin, holin and LuxR; and LuxR promoter as the promoter for anti-holin gene in E.coli, the bacteria will not survive without AHL. We clone the LuxI gene to the plasmid in Dictyostelium discoideum, making the amoeba to produce AHL. By this way, when E.coli stay in the amoeba, the AHL will be sensed by LuxR, maintaining the transcription of anti-holin gene, preventing the E.coli from lysing. But when the E.coli leave the amoeba, there will be no AHL so no anti-holin will be produced and the E.coli will be kill by endolysin.

As for constraining the Soil Cleaner in the contaminated area, a death switch is designed to restrict amoeba from migrating to nature. The kill-switch has two man-made operons. The first one includes zinTp, this promoter activity is induce by cadmium ion; and its constructive gene is lacI. The other one includes BBa_R0010, the promoter whose activity is inhibited by the protein lacI; and the constructive gene aifA and HlyA. AifA is a protein that cause the apoptosis of Dictyostelium discoideum cell. HlyA is a tag that stick after the target protein. With this tag, E.coli will secrete the target protein. When cadmium ions exist, zinTp works and lacI is generated, so the expression of aifA and HlyA is repressed. If there is no cadmium ions, BBa_R0010 will activate the expression of aifA and HlyA, so the protein aifA will be secrete right into the cytoplasm of amoeba, causing amoeba apoptosis.

Besides, instead of using traditional endogenous plasmids to transformed cyanobacteria, we cloned our biobricks into vectors that will recombine with homologous genomic DNA alternatively. Thus, horizontal gene transfer is avoided and the security and safety of our project is improved.