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                     <li>For the same reasons above, there would be minimum chance of the organisms causing any great harm to the environment. Our <i>E. coli</i> transformants do carry an ampicillin resistant gene, so accidental release of the bacteria might result in a spread of the antibiotic resistant phenotype. However, all our experiments are conducted under safe procedures and the equipments used for bacteria cells and/or cultures are properly autoclaved. </li>

         When faced with an increasing osmaolarity of the environment, the HOG pathway is activated in yeasts and the final result is the accumulation of glycerol in yeast cells to balance the exterior osmotic pressure. Sensors of the ambient osmolarity rise activates a MAPK cascade and eventually leads to the phosphorylation and activation of the Hog1 protein. Activated Hog1 then translocates into the nucleus and activates a number of transcriptional factors via protein-protein interactions or phosphorylation. These transcriptional factors (mostly activators) then mediate the expression of hundreds of genes related to cell integrity and adaptation to osmostress. Among these, the GPD1 gene and the STL1 gene are the most significant targets of the HOG pathway. GPD1 encodes the sequences for NAD-dependent glycerol-3-phosphate dehydrogenase, which is the key enzyme of glycerol sythesis. Following activation of the HOG pathway, activated Hog1 binds to the transcriptional acitvator Hot1 and upregulates the expression of GPD1. On the other hand, STL1 codes for a glycerol proton symporter in the plasma membrane of S. cerevisiae. Upon sensing a rise in osmolarity, STL1 is strongly and transiently induced by transcriptional activators Hot1 and Smp1, both members of the HOG pathway. Smp1 is phosphorylated and activated by the active Hog1 protein. Thus, the sensing of osmolarity and the induction of GPD1 and STL1 expression will make up mainly the fist part of our kill switch.<br/>

         &nbsp&nbsp&nbsp&nbsp&nbspIn order to complete our kill switch so that increasing osmolarity not only activates the HOG pathway, but also leads to cell death, we further integrate a kill gene following the promoter sequence of GPD1 and STL1. The most suitable genes would be those encoding proteins that have nuclease activity. A couple of chosen examples are NUC1 (encoding endonuclease G) and YBL055C (encoding Tat-D nuclease). Endonuclease G is the major mitochondrial nuclease in S. cerevisiae, and it induces apoptosis in yeast independently of metacaspase or of apoptosis inducing factors. Tat-D is an endo-/exo-nuclease that incises the double stranded DNA without obvious specificity via its endonuclease activity and excises the DNA from 3' to 5' end by its exonuclease activity. These proteins are intrinsically expressed during apoptosis, and their induction via osmosensitive promoters would cause irreversible harm to the yeasts and in the end kill them.<br/>

         &nbsp&nbsp&nbsp&nbsp&nbspAccording to data from current milkfish farms in Taiwan, which are saltwater farms, water osmolarity is way higher than yeast culturing environments. Therefore the HOG pathway would surely be activated once the yeasts escape from the thermogenic device, with following cell death. If the device is to be used in a fish farm with fresh water, the osmolarity would very likely be lower than the yeast culture. In light of this possibility, we are also looking into another mechanism of S. cerevisiae that is used when it is subjected to low osmolarity stress. It is called the cell integrity pathway, and is activated upon decreasing osmolarity of the environment. We hope to find similar functioning effectors downstream of the pathway like we did in the HOG pathway, and integrate the activated promoters with kill genes. If succeeded, our safety design will not be restricted to saltwater fish farms.<br/></p>

         <p style="margin-left: 40px">Every student who worked in the lab have had to received a 12-hour training. The training includes lectures on the topics &#34;Principal of Biosafety&#34; and &#34;Management of Biosafety in Laboratories&#34;.</p><br/>

         <p style="margin-left: 40px">We had an discussion with the National Taiwan University Enviroment Protection and Occupational Safety and Health Center. The assessment result was that we don&#39;t need to send the &#34;Recombinant gene experiment&#34; form to the Center, due to our materials all belonging to RG1.</p><br/>

                <p>In case of an accidental release of the thermogenic yeasts from our device to the environment, we have designed a kill switch that would ideologically lead the yeasts to death under such circumstance.</p>

                <p>When faced with an increasing osmaolarity of the environment, the HOG pathway is activated in yeasts and the final result is the accumulation of glycerol in yeast cells to balance the exterior osmotic pressure. Sensors of the ambient osmolarity rise activates a MAPK cascade and eventually leads to the phosphorylation and activation of the Hog1 protein. Activated Hog1 then translocates into the nucleus and activates a number of transcriptional factors via protein-protein interactions or phosphorylation. These transcriptional factors (mostly activators) then mediate the expression of hundreds of genes related to cell integrity and adaptation to osmostress. Among these, the GPD1 gene and the STL1 gene are the most significant targets of the HOG pathway. GPD1 encodes the sequences for NAD-dependent glycerol-3-phosphate dehydrogenase, which is the key enzyme of glycerol sythesis. Following activation of the HOG pathway, activated Hog1 binds to the transcriptional acitvator Hot1 and upregulates the expression of GPD1. On the other hand, STL1 codes for a glycerol proton symporter in the plasma membrane of S. cerevisiae. Upon sensing a rise in osmolarity, STL1 is strongly and transiently induced by transcriptional activators Hot1 and Smp1, both members of the HOG pathway. Smp1 is phosphorylated and activated by the active Hog1 protein. Thus, the sensing of osmolarity and the induction of GPD1 and STL1 expression will make up mainly the fist part of our kill switch.</p>

                <p>In order to complete our kill switch so that increasing osmolarity not only activates the HOG pathway, but also leads to cell death, we further integrate a kill gene following the promoter sequence of GPD1 and STL1. The most suitable genes would be those encoding proteins that have nuclease activity. A couple of chosen examples are NUC1 (encoding endonuclease G) and YBL055C (encoding Tat-D nuclease). Endonuclease G is the major mitochondrial nuclease in S. cerevisiae, and it induces apoptosis in yeast independently of metacaspase or of apoptosis inducing factors. Tat-D is an endo-/exo-nuclease that incises the double stranded DNA without obvious specificity via its endonuclease activity and excises the DNA from 3' to 5' end by its exonuclease activity. These proteins are intrinsically expressed during apoptosis, and their induction via osmosensitive promoters would cause irreversible harm to the yeasts and in the end kill them.</p>

                <p>According to data from current milkfish farms in Taiwan, which are saltwater farms, water osmolarity is way higher than yeast culturing environments. Therefore the HOG pathway would surely be activated once the yeasts escape from the thermogenic device, with following cell death. If the device is to be used in a fish farm with fresh water, the osmolarity would very likely be lower than the yeast culture. In light of this possibility, we are also looking into another mechanism of S. cerevisiae that is used when it is subjected to low osmolarity stress. It is called the cell integrity pathway, and is activated upon decreasing osmolarity of the environment. We hope to find similar functioning effectors downstream of the pathway like we did in the HOG pathway, and integrate the activated promoters with kill genes. If succeeded, our safety design will not be restricted to saltwater fish farms.</p>