http://2012.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=Qiaop&year=&month=2012.igem.org - User contributions [en]2024-03-28T11:44:39ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:Penn/ProjectResultsTeam:Penn/ProjectResults2012-10-27T04:02:55Z<p>Qiaop: </p>
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<div class="bigbox"><br />
<b><div class="name" align="center">A Novel Therapeutic Platform</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;">What if you could combine spatial targeting and cellular targeting into the same therapeutic? This idea is unprecedented but would allow for precise targeting of specific cells within a specific area, leaving healthy tissue intact and keeping side effects to a minimum. Higher dose precision means more of the therapeutic would be used efficiently in the targeted area and the dependency on passive diffusion – and the uncertainties that comes with it – would be eliminated.</p><br />
<br />
<p style="color:black;text-indent:30px;">The 2012 Penn iGEM team has engineered a novel, <b>modular</b> platform for targeted therapeutics that employs simultaneous spatial and cellular targeting. We have achieved spatial (and temporal) targeting with a blue light-switchable transgene expression system and cellular targeting through display of an antibody-mimetic protein on the surface of E. coli for the first time. Our platform also enables more precise dose control in the targeted area through the length of blue-light exposure, which allows us to regulate effective levels of transgene expression.</p><br />
<br />
<p style="color:black;text-indent:30px;">As a proof of concept, we applied our system to the treatment of cancer, a disease in which spatial and cellular targeting are of utmost importance. We displayed a high-affinity antibody-mimetic protein that targets Human Epidermal Growth Factor Receptor 2 (HER2), a protein commonly overexpressed in cancer cells, especially in breast cancer tumors. We combined this cellular targeting with a light-activated cytotoxic protein delivery system to successfully target and kill breast cancer cells.</p><br />
</div><br />
<br />
<div style="background-color:#01256e;" align="center"><br />
<iframe width="700" height="393" style="margin-left:10px;padding:10px; background-color:#000000;"src="http://www.youtube.com/embed/0fqLD4IJMJo" frameborder="1" allowfullscreen></iframe><br />
</div> <br><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Modularity</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;">The strength in our platform lies in its modularity. Both the light-induced transgene expression system and the surface display system work and exist independently of each other on two compatible plasmids. These plasmids can be modified to meet the needs of any synthetic biologist. </p><br />
<br />
<p style="color:black;text-indent:30px;">Any gene of interest can be cloned into the light-induced transgene expression system and will then be expressed in a light-dependent and spatially controlled manner. Any targeting protein can be cloned into our surface display platform to allow cellular targeting against any desired biomarker. These modular plasmids may then be co-transformed together to create a bacteria capable of producing a desired therapeutic molecule in a spatially and cellularly targeted manner.</p><br />
<br />
<p style="color:black;text-indent:30px;">These modular components could also be extended into applications other than medical therapeutics, such as biocatalysis, manufacturing, and alternative energy. </p><br />
<br />
<br />
<b><div class="name" align="center">Generic Components</div></b><br />
<br />
<div align="center"><br />
<table width="860" cellspacing="20" style="background-color:#d7dce1;"><br />
<tr><br />
<td width="410"><img src="https://static.igem.org/mediawiki/2012/e/e9/Lightactivationmodule.gif" width="400" height="300" /><br />
</td><br />
<td width="410"><img src="https://static.igem.org/mediawiki/2012/5/5c/Surfdispmodule.gif"width = "400" height = "300" /><br />
</td><br />
</tr><br />
<tr valign="top"><br />
<td ><br />
<p style="text-align:justify;"><b>Light Activated Gene Expression Module:</b> Light is transduced into a chemical signal through the light sensor, which through a cascade of protein activation/repression results in the light-dependent downstream expression of a desired protein.</p><br />
</td><br />
<td><br />
<p style="text-align:justify;"><b>Surface Display Module:</b> A target ligand is bound by a target binder displayed on the surface of the bacterial cell through the surface display module.</p><br />
</td><br />
</tr><br />
<br />
</table></div><br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<div class="bigbox"><br />
<br />
<br />
<br />
<br />
<b><div class="name" align="center">Proof of Concept</div></b><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2012/f/f9/Full_Color_Complete_Schematic.gif" width="700" height="525" /></div><br><br />
<p style="color:black;text-indent:30px;"> For our engineered bacterial therapeutic, we chose to target breast cancer as a proof of concept using a blue light gene expression system. We chose this specific blue-light inducible gene expression system because of the well-characterized nature of the parts and the high on:off ratio.</p><br />
<br />
<p style="color:black;text-indent:30px;"> We also identified a well-characterized class of antibody mimetic proteins called designed ankryin repeat proteins, or DARPins. One DARPin in particular (H10-2-G3) was engineered to bind to the Human Epidermal Growth Factor 2 (HER2) at picomolar affinities. HER2 is overexpressed in breast cancer cells, and we had access to cell lines that overexpressed HER2 on their cell surface which we could use for binding assays. </p><br />
<p style="color:black;text-indent:30px;"> In the future, we hope to be able to replace components in both modules of our system, allowing us to utilize different wavelengths of light to induce protein expression, target different sub populations of cells, and explore orthogonal uses for the surface display module unrelated to theraputic delivery. Further testing of our theraputic proof of concept in mice may also identify new strategies for reducing host immune system responses to the bacterial therapeutic. </p><br />
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</div><br />
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<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Human Practices</div></b><br />
<br><br />
<br />
<p style="color:black;text-indent:30px;">Upon conception of this project, we realized that although hundreds of academic research projects and iGEM projects have been conducted in the realm of Health and Medicine, almost no engineered bacterial therapeutics have been brought to the clinic. We analyzed the hurdles and road ahead for bacterial synthetic biology-enabled therapeutics, compiling a thorough report with specific actions that iGEM teams in Health/Medicine can take to make their therapies more clinically tractable. This project directly informed our wet lab work, leading us to port our therapeutic system into a non-pathogenic, probiotic bacterial strain which is already used in human therapies today.</p><br />
<br />
<br />
<p style="color:black;text-indent:30px;">We hope our targeted therapeutic platform will allow other scientists and iGEM teams to target any cells they choose. In the near term, we are planning to test our cancer cell targeting/killing bacterial system in a mouse model and make a real impact on cancer research and therapy.</p><br />
</div></div>Qiaophttp://2012.igem.org/Team:Penn/ProjectResultsTeam:Penn/ProjectResults2012-10-27T03:57:48Z<p>Qiaop: </p>
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<div>{{:Team:Penn/Template/Site}}<br />
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.all{ width:1000px; margin:0 auto;}<br />
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.name{ font-size:20px;}<br />
</style><br />
<br><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">A Novel Therapeutic Platform</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;">What if you could combine spatial targeting and cellular targeting into the same therapeutic? This idea is unprecedented but would allow for precise targeting of specific cells within a specific area, leaving healthy tissue intact and keeping side effects to a minimum. Higher dose precision means more of the therapeutic would be used efficiently in the targeted area and the dependency on passive diffusion – and the uncertainties that comes with it – would be eliminated.</p><br />
<br />
<p style="color:black;text-indent:30px;">The 2012 Penn iGEM team has engineered a novel, <b>modular</b> platform for targeted therapeutics that employs simultaneous spatial and cellular targeting. We have achieved spatial (and temporal) targeting with a blue light-switchable transgene expression system and cellular targeting through display of an antibody-mimetic protein on the surface of E. coli for the first time. Our platform also enables more precise dose control in the targeted area through the length of blue-light exposure, which allows us to regulate effective levels of transgene expression.</p><br />
<br />
<p style="color:black;text-indent:30px;">As a proof of concept, we applied our system to the treatment of cancer, a disease in which spatial and cellular targeting are of utmost importance. We displayed a high-affinity antibody-mimetic protein that targets Human Epidermal Growth Factor Receptor 2 (HER2), a protein commonly overexpressed in cancer cells, especially in breast cancer tumors. We combined this cellular targeting with a light-activated cytotoxic protein delivery system to successfully target and kill breast cancer cells.</p><br />
</div><br />
<br />
<div style="background-color:#01256e;" align="center"><br />
<iframe width="700" height="393" style="margin-left:10px;padding:10px; background-color:#000000;"src="http://www.youtube.com/embed/0fqLD4IJMJo" frameborder="1" allowfullscreen></iframe><br />
</div> <br><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Modularity</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;">The strength in our platform lies in its modularity. Both the light-induced transgene expression system and the surface display system work and exist independently of each other on two compatible plasmids. These plasmids can be modified to meet the needs of any synthetic biologist. </p><br />
<br />
<p style="color:black;text-indent:30px;">Any gene of interest can be cloned into the light-induced transgene expression system and will then be expressed in a light-dependent and spatially controlled manner. Any targeting protein can be cloned into our surface display platform to allow cellular targeting against any desired biomarker. These modular plasmids may then be co-transformed together to create a bacteria capable of producing a desired therapeutic molecule in a spatially and cellularly targeted manner.</p><br />
<br />
<p style="color:black;text-indent:30px;">These modular components could also be extended into applications other than medical therapeutics, such as biocatalysis, manufacturing, and alternative energy. </p><br />
<br />
<br />
<b><div class="name" align="center">Generic Components</div></b><br />
<br />
<div align="center"><br />
<table width="860" cellspacing="20" style="background-color:#d7dce1;"><br />
<tr><br />
<td width="410"><img src="https://static.igem.org/mediawiki/2012/e/e9/Lightactivationmodule.gif" width="400" height="300" /><br />
</td><br />
<td width="410"><img src="https://static.igem.org/mediawiki/2012/5/5c/Surfdispmodule.gif"width = "400" height = "300" /><br />
</td><br />
</tr><br />
<tr valign="top"><br />
<td ><br />
<p style="text-align:justify;"><b>Light Activated Gene Expression Module:</b> Light is transduced into a chemical signal through the light sensor, which through a cascade of protein activation/repression results in the light-dependent downstream expression of a desired protein.</p><br />
</td><br />
<td><br />
<p style="text-align:justify;"><b>Surface Display Module:</b> A target ligand is bound by a target binder displayed on the surface of the bacterial cell through the surface display module.</p><br />
</td><br />
</tr><br />
<br />
</table></div><br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<div class="bigbox"><br />
<br />
<br />
<br />
<br />
<b><div class="name" align="center">Proof of Concept</div></b><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2012/f/f9/Full_Color_Complete_Schematic.gif" width="700" height="525" /></div><br><br />
<p style="color:black;text-indent:30px;"> For our engineered bacterial therapeutic, we chose to target breast cancer as a proof of concept using a blue light gene expression system. We chose this specific blue-light inducible gene expression system because of the well-characterized nature of the parts and the high on:off ratio.</p><br />
<br />
<p style="color:black;text-indent:30px;"> We also identified a well-characterized class of antibody mimetic proteins called designed ankryin repeat proteins, or DARPins. One DARPin in particular (H10-2-G3) was engineered to bind to the Human Epidermal Growth Factor 2 (HER2) at picomolar affinities. HER2 is overexpressed in breast cancer cells, and we had access to cell lines that overexpressed HER2 on their cell surface which we could use for binding assays. </p><br />
<br />
</div><br />
<br />
<br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Human Practices</div></b><br />
<br><br />
<br />
<p style="color:black;text-indent:30px;">Upon conception of this project, we realized that although hundreds of academic research projects and iGEM projects have been conducted in the realm of Health and Medicine, almost no engineered bacterial therapeutics have been brought to the clinic. We analyzed the hurdles and road ahead for bacterial synthetic biology-enabled therapeutics, compiling a thorough report with specific actions that iGEM teams in Health/Medicine can take to make their therapies more clinically tractable. This project directly informed our wet lab work, leading us to port our therapeutic system into a non-pathogenic, probiotic bacterial strain which is already used in human therapies today.</p><br />
<br />
<br />
<p style="color:black;text-indent:30px;">We hope our targeted therapeutic platform will allow other scientists and iGEM teams to target any cells they choose. In the near term, we are planning to test our cancer cell targeting/killing bacterial system in a mouse model and make a real impact on cancer research and therapy.</p><br />
</div></div>Qiaophttp://2012.igem.org/Team:Penn/ProjectResultsTeam:Penn/ProjectResults2012-10-27T03:56:03Z<p>Qiaop: </p>
<hr />
<div>{{:Team:Penn/Template/Site}}<br />
<html><br />
<style type="text/css"><br />
.all{ width:1000px; margin:0 auto;}<br />
.ImageBorder {border: 3px solid #ffffff;margin: 0 0 20px 0;}<br />
.und{font-size:20px;color:white; text-align:center; margin:20px 0 20px 0;}<br />
.bigbox{width:936px; padding:30px;background:#d7dce1;border:2px solid #708090;border-radius:10px;-moz-border-radius:10px;-webkit-border-radius:10px;color:black; font-size:14px; text-align:justify; margin:0 0 20px 0;}<br />
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.pic1{ float:left; margin:0 40px 0 0; width:150px;}<br />
.name{ font-size:20px;}<br />
</style><br />
<br><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">A Novel Therapeutic Platform</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;">What if you could combine spatial targeting and cellular targeting into the same therapeutic? This idea is unprecedented but would allow for precise targeting of specific cells within a specific area, leaving healthy tissue intact and keeping side effects to a minimum. Higher dose precision means more of the therapeutic would be used efficiently in the targeted area and the dependency on passive diffusion – and the uncertainties that comes with it – would be eliminated.</p><br />
<br />
<p style="color:black;text-indent:30px;">The 2012 Penn iGEM team has engineered a novel, <b>modular</b> platform for targeted therapeutics that employs simultaneous spatial and cellular targeting. We have achieved spatial (and temporal) targeting with a blue light-switchable transgene expression system and cellular targeting through display of an antibody-mimetic protein on the surface of E. coli for the first time. Our platform also enables more precise dose control in the targeted area through the length of blue-light exposure, which allows us to regulate effective levels of transgene expression.</p><br />
<br />
<p style="color:black;text-indent:30px;">As a proof of concept, we applied our system to the treatment of cancer, a disease in which spatial and cellular targeting are of utmost importance. We displayed a high-affinity antibody-mimetic protein that targets Human Epidermal Growth Factor Receptor 2 (HER2), a protein commonly overexpressed in cancer cells, especially in breast cancer tumors. We combined this cellular targeting with a light-activated cytotoxic protein delivery system to successfully target and kill breast cancer cells.</p><br />
</div><br />
<br />
<div style="background-color:#01256e;" align="center"><br />
<iframe width="700" height="393" style="margin-left:10px;padding:10px; background-color:#000000;"src="http://www.youtube.com/embed/0fqLD4IJMJo" frameborder="1" allowfullscreen></iframe><br />
</div> <br><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Modularity</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;">The strength in our platform lies in its modularity. Both the light-induced transgene expression system and the surface display system work and exist independently of each other on two compatible plasmids. These plasmids can be modified to meet the needs of any synthetic biologist. </p><br />
<br />
<p style="color:black;text-indent:30px;">Any gene of interest can be cloned into the light-induced transgene expression system and will then be expressed in a light-dependent and spatially controlled manner. Any targeting protein can be cloned into our surface display platform to allow cellular targeting against any desired biomarker. These modular plasmids may then be co-transformed together to create a bacterial therapeutic for a desired disease.</p><br />
<br />
<p style="color:black;text-indent:30px;">These modular components could also be extended into applications other than medical therapeutics, such as biocatalysis, manufacturing, and alternative energy. </p><br />
<br />
<br />
<b><div class="name" align="center">Generic Components</div></b><br />
<br />
<div align="center"><br />
<table width="860" cellspacing="20" style="background-color:#d7dce1;"><br />
<tr><br />
<td width="410"><img src="https://static.igem.org/mediawiki/2012/e/e9/Lightactivationmodule.gif" width="400" height="300" /><br />
</td><br />
<td width="410"><img src="https://static.igem.org/mediawiki/2012/5/5c/Surfdispmodule.gif"width = "400" height = "300" /><br />
</td><br />
</tr><br />
<tr valign="top"><br />
<td ><br />
<p style="text-align:justify;"><b>Light Activated Gene Expression Module:</b> Light is transduced into a chemical signal through the light sensor, which through a cascade of protein activation/repression results in the light-dependent downstream expression of a desired protein.</p><br />
</td><br />
<td><br />
<p style="text-align:justify;"><b>Surface Display Module:</b> A target ligand is bound by a target binder displayed on the surface of the bacterial cell through the surface display module.</p><br />
</td><br />
</tr><br />
<br />
</table></div><br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<div class="bigbox"><br />
<br />
<br />
<br />
<br />
<b><div class="name" align="center">Proof of Concept</div></b><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2012/f/f9/Full_Color_Complete_Schematic.gif" width="700" height="525" /></div><br><br />
<p style="color:black;text-indent:30px;"> For our engineered bacterial therapeutic, we chose to target breast cancer as a proof of concept using a blue light gene expression system. We chose this specific blue-light inducible gene expression system because of the well-characterized nature of the parts and the high on:off ratio.</p><br />
<br />
<p style="color:black;text-indent:30px;"> We also identified a well-characterized class of antibody mimetic proteins called designed ankryin repeat proteins, or DARPins. One DARPin in particular (H10-2-G3) was engineered to bind to the Human Epidermal Growth Factor 2 (HER2) at picomolar affinities. HER2 is overexpressed in breast cancer cells, and we had access to cell lines that overexpressed HER2 on their cell surface which we could use for binding assays. </p><br />
<br />
</div><br />
<br />
<br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Human Practices</div></b><br />
<br><br />
<br />
<p style="color:black;text-indent:30px;">Upon conception of this project, we realized that although hundreds of academic research projects and iGEM projects have been conducted in the realm of Health and Medicine, almost no engineered bacterial therapeutics have been brought to the clinic. We analyzed the hurdles and road ahead for bacterial synthetic biology-enabled therapeutics, compiling a thorough report with specific actions that iGEM teams in Health/Medicine can take to make their therapies more clinically tractable. This project directly informed our wet lab work, leading us to port our therapeutic system into a non-pathogenic, probiotic bacterial strain which is already used in human therapies today.</p><br />
<br />
<br />
<p style="color:black;text-indent:30px;">We hope our targeted therapeutic platform will allow other scientists and iGEM teams to target any cells they choose. In the near term, we are planning to test our cancer cell targeting/killing bacterial system in a mouse model and make a real impact on cancer research and therapy.</p><br />
</div></div>Qiaophttp://2012.igem.org/Team:Penn/ProjectResultsTeam:Penn/ProjectResults2012-10-27T03:53:37Z<p>Qiaop: </p>
<hr />
<div>{{:Team:Penn/Template/Site}}<br />
<html><br />
<style type="text/css"><br />
.all{ width:1000px; margin:0 auto;}<br />
.ImageBorder {border: 3px solid #ffffff;margin: 0 0 20px 0;}<br />
.und{font-size:20px;color:white; text-align:center; margin:20px 0 20px 0;}<br />
.bigbox{width:936px; padding:30px;background:#d7dce1;border:2px solid #708090;border-radius:10px;-moz-border-radius:10px;-webkit-border-radius:10px;color:black; font-size:14px; text-align:justify; margin:0 0 20px 0;}<br />
.smallbox{ margin:20px auto; width:600px; padding:40px;background:#d7dce1;border:2px solid #708090;border-radius:10px;-moz-border-radius:10px;-webkit-border-radius:10px; overflow:hidden; text-align:justify;}<br />
.pic1{ float:left; margin:0 40px 0 0; width:150px;}<br />
.name{ font-size:20px;}<br />
</style><br />
<br><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">A Novel Therapeutic Platform</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;">What if you could combine spatial targeting and cellular targeting into the same therapeutic? This idea is unprecedented but would allow for precise targeting of specific cells within a specific area, leaving healthy tissue intact and keeping side effects to a minimum. Higher dose precision means more of the therapeutic would be used efficiently in the targeted area and the dependency on passive diffusion – and the uncertainties that comes with it – would be eliminated.</p><br />
<br />
<p style="color:black;text-indent:30px;">The 2012 Penn iGEM team has engineered a novel, <b>modular</b> platform for targeted therapeutics that employs simultaneous spatial and cellular targeting. We have achieved spatial (and temporal) targeting with a blue light-switchable transgene expression system and cellular targeting through display of an antibody-mimetic protein on the surface of E. coli for the first time. Our platform also enables more precise dose control in the targeted area through the length of blue-light exposure, which allows us to regulate effective levels of transgene expression.</p><br />
<br />
<p style="color:black;text-indent:30px;">As a proof of concept, we applied our system to the treatment of cancer, a disease in which spatial and cellular targeting are of utmost importance. We displayed a high-affinity antibody-mimetic protein that targets Human Epidermal Growth Factor Receptor 2 (HER2), a protein commonly overexpressed in cancer cells, especially in breast cancer tumors. We combined this cellular targeting with a light-activated cytotoxic protein delivery system to successfully target and kill breast cancer cells.</p><br />
</div><br />
<br />
<div style="background-color:#01256e;" align="center"><br />
<iframe width="700" height="393" style="margin-left:10px;padding:10px; background-color:#000000;"src="http://www.youtube.com/embed/0fqLD4IJMJo" frameborder="1" allowfullscreen></iframe><br />
</div> <br><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Modularity</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;">The strength in our platform lies in its modularity. Both the light-induced transgene expression system and the surface display system work and exist independently of each other on two compatible plasmids. These plasmids can be modified to meet the needs of any synthetic biologist. </p><br />
<br />
<p style="color:black;text-indent:30px;">Any gene of interest can be cloned into the light-induced transgene expression system and will then be expressed in a light-dependent and spatially controlled manner. Any targeting protein can be cloned into our surface display platform to allow cellular targeting against any desired biomarker. These modular plasmids may then be co-transformed together to create a bacterial therapeutic for a desired disease.</p><br />
<br />
<p style="color:black;text-indent:30px;">These modular components could also be extended into applications other than medical therapeutics, such as biocatalysis, manufacturing, and alternative energy. </p><br />
<br />
<br />
<b><div class="name" align="center">Generic Components</div></b><br />
<br />
<div align="center"><br />
<table width="860" cellspacing="20" style="background-color:#d7dce1;"><br />
<tr><br />
<td width="410"><img src="https://static.igem.org/mediawiki/2012/e/e9/Lightactivationmodule.gif" width="400" height="300" /><br />
</td><br />
<td width="410"><img src="https://static.igem.org/mediawiki/2012/5/5c/Surfdispmodule.gif"width = "400" height = "300" /><br />
</td><br />
</tr><br />
<tr valign="top"><br />
<td ><br />
<p style="text-align:justify;"><b>Light Activated Gene Expression Module:</b> Light is transduced into a chemical signal through the light sensor, which through a cascade of protein activation/repression results in the light-dependent downstream expression of a desired protein.</p><br />
</td><br />
<td><br />
<p style="text-align:justify;"><b>Surface Display Module:</b> Monoclonal antibodies identify antigens on certain cells or viruses. Monoclonal antibodies are often coupled with therapeutic agents. However, if the antigen is present in healthy tissue outside the diseased area, it will be targeted as well.</p><br />
</td><br />
</tr><br />
<br />
</table></div><br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<div class="bigbox"><br />
<br />
<br />
<br />
<br />
<b><div class="name" align="center">Proof of Concept</div></b><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2012/f/f9/Full_Color_Complete_Schematic.gif" width="700" height="525" /></div><br><br />
<p style="color:black;text-indent:30px;"> For our engineered bacterial therapeutic, we chose to target breast cancer as a proof of concept using a blue light gene expression system. We chose this specific blue-light inducible gene expression system because of the well-characterized nature of the parts and the high on:off ratio.</p><br />
<br />
<p style="color:black;text-indent:30px;"> We also identified a well-characterized class of antibody mimetic proteins called designed ankryin repeat proteins, or DARPins. One DARPin in particular (H10-2-G3) was engineered to bind to the Human Epidermal Growth Factor 2 (HER2) at picomolar affinities. HER2 is overexpressed in breast cancer cells, and we had access to cell lines that overexpressed HER2 on their cell surface which we could use for binding assays. </p><br />
<br />
</div><br />
<br />
<br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Human Practices</div></b><br />
<br><br />
<br />
<p style="color:black;text-indent:30px;">Upon conception of this project, we realized that although hundreds of academic research projects and iGEM projects have been conducted in the realm of Health and Medicine, almost no engineered bacterial therapeutics have been brought to the clinic. We analyzed the hurdles and road ahead for bacterial synthetic biology-enabled therapeutics, compiling a thorough report with specific actions that iGEM teams in Health/Medicine can take to make their therapies more clinically tractable. This project directly informed our wet lab work, leading us to port our therapeutic system into a non-pathogenic, probiotic bacterial strain which is already used in human therapies today.</p><br />
<br />
<br />
<p style="color:black;text-indent:30px;">We hope our targeted therapeutic platform will allow other scientists and iGEM teams to target any cells they choose. In the near term, we are planning to test our cancer cell targeting/killing bacterial system in a mouse model and make a real impact on cancer research and therapy.</p><br />
</div></div>Qiaophttp://2012.igem.org/Team:Penn/ProjectResultsTeam:Penn/ProjectResults2012-10-27T03:52:12Z<p>Qiaop: </p>
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<div class="bigbox"><br />
<b><div class="name" align="center">A Novel Therapeutic Platform</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;">What if you could combine spatial targeting and cellular targeting into the same therapeutic? This idea is unprecedented but would allow for precise targeting of specific cells within a specific area, leaving healthy tissue intact and keeping side effects to a minimum. Higher dose precision means more of the therapeutic would be used efficiently in the targeted area and the dependency on passive diffusion – and the uncertainties that comes with it – would be eliminated.</p><br />
<br />
<p style="color:black;text-indent:30px;">The 2012 Penn iGEM team has engineered a novel, <b>modular</b> platform for targeted therapeutics that employs simultaneous spatial and cellular targeting. We have achieved spatial (and temporal) targeting with a blue light-switchable transgene expression system and cellular targeting through display of an antibody-mimetic protein on the surface of E. coli for the first time. Our platform also enables more precise dose control in the targeted area through the length of blue-light exposure, which allows us to regulate effective levels of transgene expression.</p><br />
<br />
<p style="color:black;text-indent:30px;">As a proof of concept, we applied our system to the treatment of cancer, a disease in which spatial and cellular targeting are of utmost importance. We displayed a high-affinity antibody-mimetic protein that targets Human Epidermal Growth Factor Receptor 2 (HER2), a protein commonly overexpressed in cancer cells, especially in breast cancer tumors. We combined this cellular targeting with a light-activated cytotoxic protein delivery system to successfully target and kill breast cancer cells.</p><br />
</div><br />
<br />
<div style="background-color:#01256e;" align="center"><br />
<iframe width="700" height="393" style="margin-left:10px;padding:10px; background-color:#000000;"src="http://www.youtube.com/embed/0fqLD4IJMJo" frameborder="1" allowfullscreen></iframe><br />
</div> <br><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Modularity</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;">The strength in our platform lies in its modularity. Both the light-induced transgene expression system and the surface display system work and exist independently of each other on two compatible plasmids. These plasmids can be modified to meet the needs of any synthetic biologist. </p><br />
<br />
<p style="color:black;text-indent:30px;">Any gene of interest can be cloned into the light-induced transgene expression system and will then be expressed in a light-dependent and spatially controlled manner. Any targeting protein can be cloned into our surface display platform to allow cellular targeting against any desired biomarker. These modular plasmids may then be co-transformed together to create a bacterial therapeutic for a desired disease.</p><br />
<br />
<p style="color:black;text-indent:30px;">These modular components could also be extended into applications other than medical therapeutics, such as biocatalysis, manufacturing, and alternative energy. </p><br />
<br />
<br />
<b><div class="name" align="center">Generic Components</div></b><br />
<br />
<div align="center"><br />
<table width="860" cellspacing="20" style="background-color:#d7dce1;"><br />
<tr><br />
<td width="410"><img src="https://static.igem.org/mediawiki/2012/e/e9/Lightactivationmodule.gif" width="400" height="300" /><br />
</td><br />
<td width="410"><img src="https://static.igem.org/mediawiki/2012/5/5c/Surfdispmodule.gif"width = "400" height = "300" /><br />
</td><br />
</tr><br />
<tr valign="top"><br />
<td ><br />
<p style="text-align:justify;"><b>Light Activated Gene Expression Module:</b> Surgeons excise a tumor manually, without regard for cellular heterogeneity within and around the tumor area.</p><br />
</td><br />
<td><br />
<p style="text-align:justify;"><b>Surface Display Module:</b> Monoclonal antibodies identify antigens on certain cells or viruses. Monoclonal antibodies are often coupled with therapeutic agents. However, if the antigen is present in healthy tissue outside the diseased area, it will be targeted as well.</p><br />
</td><br />
</tr><br />
<br />
</table></div><br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<div class="bigbox"><br />
<br />
<br />
<br />
<br />
<b><div class="name" align="center">Proof of Concept</div></b><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2012/f/f9/Full_Color_Complete_Schematic.gif" width="700" height="525" /></div><br><br />
<p style="color:black;text-indent:30px;"> For our engineered bacterial therapeutic, we chose to target breast cancer as a proof of concept using a blue light gene expression system. We chose this specific blue-light inducible gene expression system because of the well-characterized nature of the parts and the high on:off ratio.</p><br />
<br />
<p style="color:black;text-indent:30px;"> We also identified a well-characterized class of antibody mimetic proteins called designed ankryin repeat proteins, or DARPins. One DARPin in particular (H10-2-G3) was engineered to bind to the Human Epidermal Growth Factor 2 (HER2) at picomolar affinities. HER2 is overexpressed in breast cancer cells, and we had access to cell lines that overexpressed HER2 on their cell surface which we could use for binding assays. </p><br />
<br />
</div><br />
<br />
<br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Human Practices</div></b><br />
<br><br />
<br />
<p style="color:black;text-indent:30px;">Upon conception of this project, we realized that although hundreds of academic research projects and iGEM projects have been conducted in the realm of Health and Medicine, almost no engineered bacterial therapeutics have been brought to the clinic. We analyzed the hurdles and road ahead for bacterial synthetic biology-enabled therapeutics, compiling a thorough report with specific actions that iGEM teams in Health/Medicine can take to make their therapies more clinically tractable. This project directly informed our wet lab work, leading us to port our therapeutic system into a non-pathogenic, probiotic bacterial strain which is already used in human therapies today.</p><br />
<br />
<br />
<p style="color:black;text-indent:30px;">We hope our targeted therapeutic platform will allow other scientists and iGEM teams to target any cells they choose. In the near term, we are planning to test our cancer cell targeting/killing bacterial system in a mouse model and make a real impact on cancer research and therapy.</p><br />
</div></div>Qiaophttp://2012.igem.org/Team:Penn/AchievementsTeam:Penn/Achievements2012-10-27T03:39:18Z<p>Qiaop: </p>
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</style><br />
<br><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Modular Light Dependent Expression of Cytolysin A</div></b><br />
<br><br />
<p style="text-align:justify;"><br />
We are the first to utilize the pDawn YF1/FixJ system to express the ClyA hemolysin protein as a drug delivery system. We have shown that the system is capable of responding to blue light stimulus in a relatively short time span, and that the expression is entirely light dependent and is tightly controlled. We have further shown that this module is capable of operation independent of the remainder of the system, and that the pDawn system is capable of operating in non-traditional <i>E. coli.</i> strains such as Nissle 1917. Furthermore, due to the interchangeability of various parts in the module, the behavior of the system can be easily altered and re-purposed for other tasks unrelated to drug delivery.<br />
</p><br />
</div><br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Modular Surface Display System</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
We are the first to successfully utilize the INPNC membrane-bound protein to display DARPin H10-2-G3 that retains its picomolar affinity for HER2. As a result of these experiments, we have developed a INPNC surface display vector, which allows for the simple and straightforward display of a wide variety of proteins such as fluorescent proteins (mCherry, antibody mimetic proteins such as DARPin H10-2-G3, and epitope tags such as the Human influenza hemagglutinin (HA) tag. This display vector has been show to work independently of the complete proposed system. Furthermore, due to the interchangeability of various parts in the module, the behavior of the system can be easily altered and re-purposed for other tasks unrelated to drug delivery.<br />
</p><br />
</div><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">VerifiGEM: A New Way to Test Biobricks</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
Our team proposed a new framework for distributing the monumental task of Biobrick verification and quality control, a process currently undertaken solely by iGEM HQ. We developed a user interface and organizational schema that would enable teams to quickly and efficiently test each others' biobricks, provide feedback, and form an ad-hoc network of laboratories that can better handle the "rush" of biobricks produced during the iGEM competition.<br />
</div><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">A Consideration of the Bench to Bedside Transition of Bacterial Therapeutics</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
We performed an in depth investigation of the complex interaction between scientists and the public to explain the lack of bacterial therapeutics in the current drug development pipeline. We provided guidelines for how iGEM teams can act to further the progress of bacterial therapeutics and synthetic biology in general. As a result of our research, we performed additional experiments, providing an example of how careful experimental design can help research teams better anticipate and navigate the drug development process.<br />
</div><br />
</body><br />
</html></div>Qiaophttp://2012.igem.org/Team:Penn/AchievementsTeam:Penn/Achievements2012-10-27T03:36:41Z<p>Qiaop: </p>
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<br><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Modular Light Dependent Expression of Cytolysin A</div></b><br />
<br><br />
<p style="text-align:justify;"><br />
We are the first to utilize the pDawn YF1/FixJ system to express the ClyA hemolysin protein as a drug delivery system. We have shown that the system is capable of responding to blue light stimulus in a relatively short time span, and that the expression is entirely light dependent and is tightly controlled. We have further shown that this module is capable of operation independent of the remainder of the system, and that the pDawn system is capable of operating in non-traditional <i>E. coli.</i> strains such as Nissle 1917. <br />
</p><br />
</div><br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Modular Surface Display System</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
We are the first to successfully utilize the INPNC membrane-bound protein to display DARPin H10-2-G3 that retains its picomolar affinity for HER2. As a result of these experiments, we have developed a INPNC surface display vector, which allows for the simple and straightforward display of a wide variety of proteins such as fluorescent proteins (mCherry, antibody mimetic proteins such as DARPin H10-2-G3, and epitope tags such as the Human influenza hemagglutinin (HA) tag.<br />
</p><br />
</div><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">VerifiGEM: A New Way to Test Biobricks</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
Our team proposed a new framework for distributing the monumental task of Biobrick verification and quality control, a process currently undertaken solely by iGEM HQ. We developed a user interface and organizational schema that would enable teams to quickly and efficiently test each others' biobricks, provide feedback, and form an ad-hoc network of laboratories that can better handle the "rush" of biobricks produced during the iGEM competition.<br />
</div><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">A Consideration of the Bench to Bedside Transition of Bacterial Therapeutics</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
We performed an in depth investigation of the complex interaction between scientists and the public to explain the lack of bacterial therapeutics in the current drug development pipeline. We provided guidelines for how iGEM teams can act to further the progress of bacterial therapeutics and synthetic biology in general. As a result of our research, we performed additional experiments, providing an example of how careful experimental design can help research teams better anticipate and navigate the drug development process.<br />
</div><br />
</body><br />
</html></div>Qiaophttp://2012.igem.org/Team:Penn/AchievementsTeam:Penn/Achievements2012-10-27T03:27:47Z<p>Qiaop: </p>
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<br><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Modular Light Dependent Expression of Cytolysin A</div></b><br />
<br><br />
<p style="text-align:justify;"><br />
We are the first to utilize the pDawn YF1/FixJ system to express the ClyA hemolysin protein as a drug delivery system. We have shown that the system is capable of responding to blue light stimulus in a relatively short time span, and that the expression is entirely light dependent and is tightly controlled. We have further shown that this module is capable of operation independent of the remainder of the system, and that the pDawn system is capable of operating in non-traditional <i>E. coli.</i> strains such as Nissle 1917. <br />
</p><br />
</div><br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Modular Surface Display System</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
We are the first to successfully utilize the INPNC membrane-bound protein to display DARPin H10-2-G3 that retains its picomolar affinity for HER2. As a result of these experiments, we have developed a INPNC surface display vector, which allows for the simple and straightforward display of a wide variety of proteins such as fluorescent proteins (mCherry, antibody mimetic proteins such as DARPin H10-2-G3, and epitope tags such as the Human influenza hemagglutinin (HA) tag.<br />
</p><br />
</div><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Perception Barriers to Bacterial Therapeutics</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
However, the removal of biological barriers to bacterial therapeutics alone is not sufficient to enable bacterial therapeutics to move into the drug development pipeline. Through the course of recent history, many high profile technologies, such as gene therapy or nanotechnology have been met with public skepticism and even fear, as the technologies failed to deliver on earlier promises. This prompted a constriction of available funding and subsequently impeded progress in those fields, an outcome that nanotechnology in particular is only just beginning to recover from.<br />
</p><br />
<p style="color:black;text-indent:30px;"><br />
We propose a model, adapted from Gartner Inc, which proposes that the disconnect between the expectations of the public and the realities of scientific research produces an initial "peak of inflated expectations" (and funding), that rapidly disappears as promised advances are delayed or do not work as planned (Figure 1). We believe that this "trough of disillusionment" is the cause of restrictions in funding and a general stall of scientific progress in a given field. We propose that the peak can be smoothed out, reducing the size of peak, but more importantly, eliminating the trough of disillusionment (Figure 2).<br />
</p><br />
<br />
<div class="figs2"><br />
<div class="fignew"><div align="center"><img src="https://static.igem.org/mediawiki/2012/7/7c/Hype-Cycle-Original.jpg" height="200" width="320"><br><br />
<b>Figure 1</b></div><div style="text-align:center;">Figure 1: The hype cycle without the modulating effects of public outreach efforts.</div></div><br><br />
<br />
<div class="fignew"><div align="center"><img src="https://static.igem.org/mediawiki/2012/a/ab/Hype-Cycle-Original---Copy.jpg" height="200" width="320"><br><br />
<b>Figure 2</b></div><div style="text-align:center;">Figure 2: The modulation of the extremes of the hype cycle through public outreach efforts such as iGEM. </div></div></div><br><br />
<br />
<br />
<p style="color:black;text-indent:30px;"><br />
What then, must we as synthetic biologists do to prevent this fate from befalling our own field of study? Certainly, scientists must perform a balancing act between reporting the advances they have made and making realistic conclusions. iGEM teams in particular have a unique opportunity to impact this cycle. Each time a team teaches a class to younger students, presents their research, or even sets up a fun experiment at a local science event, they are given an opportunity to communicate the potential and the limitations of synthetic biology. This is an opportunity many researchers do not have, and should be treated as more than simply a time to take pictures and have fun (although those are certainly important parts of these events nevertheless). <br />
</p><br />
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</body><br />
</html></div>Qiaophttp://2012.igem.org/Team:Penn/AchievementsTeam:Penn/Achievements2012-10-27T03:27:30Z<p>Qiaop: </p>
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.name{ font-size:20px;}<br />
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<br><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Modular Light Dependent Expression of Cytolysin A</div></b><br />
<br><br />
<p style="text-align:justify;"><br />
We are the first to utilize the pDawn YF1/FixJ system to express the ClyA hemolysin protein as a drug delivery system. We have shown that the system is capable of responding to blue light stimulus in a relatively short time span, and that the expression is entirely light dependent and is tightly controlled. We have further shown that this module is capable of operation independent of the remainder of the system, and that the pDawn system is capable of operating in non-traditional <i>E. coli.</i> strains such as Nissle 1917. <br />
</p><br />
</div><br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Modular Surface Display System</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
We are the first to successfully utilize the INPNC membrane-bound protein to display DARPin H10-2-G3 that retains its picomolar affinity for HER2. As a result of these experiments, we have developed a INPNC surface display vector, which allows for the simple and straightforward display of a wide variety of proteins such as fluorescent proteins (mCherry, antibody mimetic proteins such as DARPin H10-2-G3, and epitope tags such as the Human influenza hemagglutinin (HA) tag.<br />
</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/c/c6/VerifiGEM-Logo.jpg/800px-VerifiGEM-Logo.jpg" width="900" height="200" /></div><br />
<p style="color:black;text-indent:30px;"><br />
Furthermore, in an effort to speed progress and information sharing between iGEM teams, government research organizations, and private research groups, we have proposed a system known as VerifiGEM that would allow for the quality control of BioBricks to be distributed across the entire iGEM community. Should our system be implemented, the overall quality and reliability of BioBricks will be improved greatly, at very little cost to individual iGEM teams. <br />
</p><br />
</div><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Perception Barriers to Bacterial Therapeutics</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
However, the removal of biological barriers to bacterial therapeutics alone is not sufficient to enable bacterial therapeutics to move into the drug development pipeline. Through the course of recent history, many high profile technologies, such as gene therapy or nanotechnology have been met with public skepticism and even fear, as the technologies failed to deliver on earlier promises. This prompted a constriction of available funding and subsequently impeded progress in those fields, an outcome that nanotechnology in particular is only just beginning to recover from.<br />
</p><br />
<p style="color:black;text-indent:30px;"><br />
We propose a model, adapted from Gartner Inc, which proposes that the disconnect between the expectations of the public and the realities of scientific research produces an initial "peak of inflated expectations" (and funding), that rapidly disappears as promised advances are delayed or do not work as planned (Figure 1). We believe that this "trough of disillusionment" is the cause of restrictions in funding and a general stall of scientific progress in a given field. We propose that the peak can be smoothed out, reducing the size of peak, but more importantly, eliminating the trough of disillusionment (Figure 2).<br />
</p><br />
<br />
<div class="figs2"><br />
<div class="fignew"><div align="center"><img src="https://static.igem.org/mediawiki/2012/7/7c/Hype-Cycle-Original.jpg" height="200" width="320"><br><br />
<b>Figure 1</b></div><div style="text-align:center;">Figure 1: The hype cycle without the modulating effects of public outreach efforts.</div></div><br><br />
<br />
<div class="fignew"><div align="center"><img src="https://static.igem.org/mediawiki/2012/a/ab/Hype-Cycle-Original---Copy.jpg" height="200" width="320"><br><br />
<b>Figure 2</b></div><div style="text-align:center;">Figure 2: The modulation of the extremes of the hype cycle through public outreach efforts such as iGEM. </div></div></div><br><br />
<br />
<br />
<p style="color:black;text-indent:30px;"><br />
What then, must we as synthetic biologists do to prevent this fate from befalling our own field of study? Certainly, scientists must perform a balancing act between reporting the advances they have made and making realistic conclusions. iGEM teams in particular have a unique opportunity to impact this cycle. Each time a team teaches a class to younger students, presents their research, or even sets up a fun experiment at a local science event, they are given an opportunity to communicate the potential and the limitations of synthetic biology. This is an opportunity many researchers do not have, and should be treated as more than simply a time to take pictures and have fun (although those are certainly important parts of these events nevertheless). <br />
</p><br />
</div><br />
</body><br />
</html></div>Qiaophttp://2012.igem.org/Team:Penn/AchievementsTeam:Penn/Achievements2012-10-27T03:22:36Z<p>Qiaop: </p>
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<b><div class="name" align="center">Modular Light Dependent Expression of Proteins</div></b><br />
<br><br />
<p style="text-align:justify;"><br />
We are the first to utilize the pDawn YF1/FixJ system to express the ClyA hemolysin protein as a drug delivery system. We have shown that the system is capable of responding to blue light stimulus in a relatively short time span, and that the expression is entirely light dependent and is tightly controlled. We have further shown that this module is capable of operation independent of the remainder of the system, and that the pDawn system is capable of operating in non-traditional <i>E. coli.</i> strains such as Nissle 1917. <br />
</p><br />
</div><br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Biological Barriers to Bacterial Therapeutics</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
From a technological standpoint, there is a great deal of work that remains to be done before a bacterial therapeutic can enter the drug development pipeline. While many iGEM teams, including us, have helped set the groundwork for bacterial therapeutics, there are still some biological barriers to a bacterial therapeutic. We identified the immunogenicity of laboratory strains of <i>E. coli.</i> as a major biological barrier. We then investigated methods to decrease the immunogenicity of <i>E. coli.</i>, eventually choosing to port modules of our target drug delivery system into a non-immunogenic strain of <i>E. coli.</i>, Nissle 1917. <br />
</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/c/c6/VerifiGEM-Logo.jpg/800px-VerifiGEM-Logo.jpg" width="900" height="200" /></div><br />
<p style="color:black;text-indent:30px;"><br />
Furthermore, in an effort to speed progress and information sharing between iGEM teams, government research organizations, and private research groups, we have proposed a system known as VerifiGEM that would allow for the quality control of BioBricks to be distributed across the entire iGEM community. Should our system be implemented, the overall quality and reliability of BioBricks will be improved greatly, at very little cost to individual iGEM teams. <br />
</p><br />
</div><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Perception Barriers to Bacterial Therapeutics</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
However, the removal of biological barriers to bacterial therapeutics alone is not sufficient to enable bacterial therapeutics to move into the drug development pipeline. Through the course of recent history, many high profile technologies, such as gene therapy or nanotechnology have been met with public skepticism and even fear, as the technologies failed to deliver on earlier promises. This prompted a constriction of available funding and subsequently impeded progress in those fields, an outcome that nanotechnology in particular is only just beginning to recover from.<br />
</p><br />
<p style="color:black;text-indent:30px;"><br />
We propose a model, adapted from Gartner Inc, which proposes that the disconnect between the expectations of the public and the realities of scientific research produces an initial "peak of inflated expectations" (and funding), that rapidly disappears as promised advances are delayed or do not work as planned (Figure 1). We believe that this "trough of disillusionment" is the cause of restrictions in funding and a general stall of scientific progress in a given field. We propose that the peak can be smoothed out, reducing the size of peak, but more importantly, eliminating the trough of disillusionment (Figure 2).<br />
</p><br />
<br />
<div class="figs2"><br />
<div class="fignew"><div align="center"><img src="https://static.igem.org/mediawiki/2012/7/7c/Hype-Cycle-Original.jpg" height="200" width="320"><br><br />
<b>Figure 1</b></div><div style="text-align:center;">Figure 1: The hype cycle without the modulating effects of public outreach efforts.</div></div><br><br />
<br />
<div class="fignew"><div align="center"><img src="https://static.igem.org/mediawiki/2012/a/ab/Hype-Cycle-Original---Copy.jpg" height="200" width="320"><br><br />
<b>Figure 2</b></div><div style="text-align:center;">Figure 2: The modulation of the extremes of the hype cycle through public outreach efforts such as iGEM. </div></div></div><br><br />
<br />
<br />
<p style="color:black;text-indent:30px;"><br />
What then, must we as synthetic biologists do to prevent this fate from befalling our own field of study? Certainly, scientists must perform a balancing act between reporting the advances they have made and making realistic conclusions. iGEM teams in particular have a unique opportunity to impact this cycle. Each time a team teaches a class to younger students, presents their research, or even sets up a fun experiment at a local science event, they are given an opportunity to communicate the potential and the limitations of synthetic biology. This is an opportunity many researchers do not have, and should be treated as more than simply a time to take pictures and have fun (although those are certainly important parts of these events nevertheless). <br />
</p><br />
</div><br />
</body><br />
</html></div>Qiaophttp://2012.igem.org/Team:Penn/TeamTeam:Penn/Team2012-10-27T03:10:14Z<p>Qiaop: </p>
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<div style="text-align:center;font-size:34px;color:white;"><b>Our Team</b></div><br><br />
<br />
<div class="bigbox"><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2012/7/7a/Penn-iGEM-2012-Team-Photo.jpg" height="500" width="700" /></div><br><br />
The Penn iGEM 2012 Team consists of 4 undergraduates, 3 advisors, and many others who have provided important contributions along the way. Together, we have learned a lot over the last few months. We have taught ourselves different protocols and developed new standards to synchronize our work. Through our participation in iGEM, we have collaborated to learn more about synthetic biology - from the initial days of cloning constructs to the final days of imaging and analysis. Our idea for spatio-temporal control of drug delivery first originated in late May after weeks of reading papers. Slowly we have been able to piece together different components of the system to help the project materialize to the system it is today. We believe our work in optogenetics and drug delivery has a promising future and we are excited to share our results at the World Championships! <br />
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<br />
<div class="und">Undergraduates</div><br />
<br />
<br />
<div class="smallbox"><div class="flpic"><br />
<img src="https://static.igem.org/mediawiki/2012/c/c6/Clark_Park_4.jpg" class="pic1" /></div><div class="flleft"><br />
<div style="text-align:center;"><b class="name">Ashwin Amurthur</b><br />
<br><br />
<i>"Now we're rolling"</i><br></div><br />
<br><br />
Ashwin is a sophomore at the University of Pennsylvania studying bioengineering and management.<br />
<br><br><br />
<b>Natural lab habitat:</b> On the confocal<br><br />
<b>Best kept lab secret:</b> Never ran a PCR <br><br />
<b>Favorite Experiment Celebration GIF:<a href="http://tinyurl.com/9mbudvt">http://tinyurl.com/9mbudvt</a></b> <br />
</div></div><br />
<br />
<br />
<div class="smallbox"><div class="flpic"><br />
<img src="http://benjaminshyong.com/igem2011/wp-content/uploads/2011/09/283077_1448493822523_1538520160_31353808_6931705_n-e1317115984293-221x300.jpg" class="pic1" /></div><div class="flleft"><br />
<div style="text-align:center;"><b class="name">Michael Magaraci</b><br />
<br><i>"Sorry guys, I overslept..."</i><br></div><br><br />
Mike is a senior at the University of Pennsylvania studying bioengineering and management. <br />
<br><br><br />
<b>Natural lab habitat:</b> At the PCR machine<br><br />
<b>Best kept lab secret:</b> <br><br />
<b>Favorite Experiment Celebration GIF:<a href="http://tinyurl.com/9rpe7ap">http://tinyurl.com/9rpe7ap</a></b> <br />
</div></div><br />
<br />
<div class="smallbox"><div class="flpic"><br />
<img src="http://benjaminshyong.com/igem2011/wp-content/uploads/2011/09/Untitled-e1317089819600.png" class="pic1" /></div><div class="flleft"><br />
<div style="text-align:center;"><b class="name">Peter Qiao</b><br />
<br><i>"There's nothing mini about my miniprep yields"</i><br></div><br><br />
Peter is a junior at the University of Pennsylvania studying bioengineering. He hopes to pursue an M.D/Ph.D in the future.<br />
<br><br><br />
<b>Natural lab habitat:</b> In the hood passaging cells<Br><br />
<b>Best kept lab secret:</b> Triple ligation <br><br />
<b>Favorite Experiment Celebration GIF: <a href="http://tinyurl.com/8e2zj3l">http://tinyurl.com/8e2zj3l</a></b> <br />
<br />
</div></div><br />
<br />
<div class="smallbox"><div class="flpic"><br />
<img src="http://benjaminshyong.com/igem2011/wp-content/uploads/2011/09/P1000543-e1317095308771-221x300.jpg" class="pic1" /></div><div class="flleft"><br />
<div style="text-align:center;"><b class="name">Avin Veerakumar</b><br />
<br><i>"Do you want to sleep or do you want to win?"</i><br></div><br><br />
Avin is a senior at the University of Pennsylvania studying Bioengineering and Management.<br />
<br><br><br />
<b>Natural lab habitat:</b> On the confocal<br><br />
<b>Best kept lab secret:</b> Never ran a PCR <br><br />
<b>Favorite Experiment Celebration GIF: <a href="http://tinyurl.com/9ahlsc4">http://tinyurl.com/9ahlsc4</a></b> <br />
</div></div><br />
<br />
<div class="und">Advisors</div><br />
<br />
<div class="smallbox"><div class="flpic"><br />
<img src="http://benjaminshyong.com/igem2011/wp-content/uploads/2011/09/sarkar_web-e1317090202691.jpg" class="pic1" /></div><div class="flleft"><br />
<b class="name">Dr. Casim A. Sarkar</b><br />
<br><br><br />
Dr. Casim A. Sarkar is the Principal Investigator of the Molecular Cell Engineering Laboratory at the University of Pennsylvania Department of Bioengineering. His research interests include molecular cell engineering, protein engineering, ligand/receptor binding and trafficking, cell signaling and decision making, and computational, synthetic, and systems biology. Dr. Sarkar received a PhD in Chemical Engineering with a minor in Computational Biology at MIT and a BS in Chemical Engineering at the University of Texas at Austin. <br />
</div></div><br />
<div class="smallbox"><div class="flpic"><br />
<img src="http://www.med.upenn.edu/apps/my/images/faculty_pics/goul2044.jpg" class="pic1" /></div><div class="flleft"><br />
<b class="name">Dr. Mark Goulian</b><br />
<br><br><br />
Dr. Mark Goulian is the Edmund J. and Louise W. Kahn Endowed Term Professor of Biology at the University of Pennsylvania. His research is focused on the regulatory circuits that bacteria use to sense and respond to the environment. His other research interests include two-component signaling in E. coli and directed evolution of signaling circuits. Dr. Goulian received his PhD from Harvard University. </div><br />
</div><br />
<div class="smallbox"><div class="flpic"><br />
<img src="http://benjaminshyong.com/igem2011/wp-content/uploads/2011/09/MillerJordan_Penn-Fellow-e1317190345626.jpg" class="pic1" /></div><div class="flleft"><br />
<b class="name">Dr. Jordan S. Miller</b><br />
<br><br><br />
Dr. Jordan S. Miller is a postdoctoral fellow at the University of Pennsylvania in Dr. Christopher S. Chen's Tissue Microfabrication Laboratory in the Department of Bioengineering. He is currently also a board member of Hive76 and before his time at Penn, he was a developer at RepRap and and an associate at PTV Sciences. Dr. Miller received a PhD from Rice University and earned his undergraduate degree from MIT.<br />
</div></div><br />
<div class="und">Acknowledgements</div><br />
<div class="smallbox"><div class="flpic"><br />
<img src="http://benjaminshyong.com/igem2011/wp-content/uploads/2011/09/DaphneNg-e1317243791521-224x300.jpg" class="pic1" /></div><div class="flleft"><br />
<b class="name">Daphne Ng</b><br />
<br><br><br />
Daphne Ng is a PhD candidate at the University of Pennsylvania in Dr. Casim A. Sarkar's Molecular Cell Engineering Laboratory in the Department of Bioengineering. She graduated from Cornell University in 2008 with a B.S. in Chemical Engineering. The 2012 Penn iGEM team would like to thank Daphne for her contribution to the cloning and design process, as well as providing expression and cloning vectors.</div><br />
</div><br />
<div class="smallbox"><div class="flpic"><br />
<img src="http://benjaminshyong.com/igem2011/wp-content/uploads/2011/09/najafshah.jpg" class="pic1" /></div><div class="flleft"><br />
<b class="name">Najaf A. Shah</b><br />
<br><br><br />
Najaf Shah is a PhD candidate at the University of Pennsylvania in Dr. Casim A. Sarkar's Molecular Cell Engineering Laboratory in the Department of Bioengineering. The 2012 Penn iGEM team would like to thank Najaf for providing input on cloning, input on system design, and access to advanced imaging facilities.</div><br />
</div><br />
<div class="smallbox"><div class="flpic"><br />
<img src="http://benjaminshyong.com/igem2011/wp-content/uploads/2011/09/board-sevile.jpg" class="pic1" /></div><div class="flleft"><br />
<b class="name">Sevile Mannickarottu</b><br />
<br><br><br />
Sevile Mannickarottu is the Director of Bioengineering Instructional Laboratories at the University of Pennsylvania. Before his current post, he was the Philadelphia Operations Manager for Technology Education Awareness and a Project Electrical Engineer at Lutron Electronics. He received an MLA in Religious Studies and a BSc in Electric Engineering from the University of Pennsylvania and is currently pursuing a PhD in Religious Studies. The 2012 Penn iGEM team would like to thank Sevile for providing his facilities and equipment for use during the project.<br />
</div></div><br />
<div class="und">Contributions</div><br />
<br />
<div class="bigbox"><br />
The Penn iGEM team would also like to thank the following individuals for their significant contributions to the team this year:<br />
<ul style="font-size:17px;padding-left:20px; margin:0 0;"><br />
<li><b>Christopher Fang-Yen</b>, PhD, Assistant Professor of Bioengineering at University of Pennsylvania</li><br />
<li><b>Matthew Lazzara</b>, PhD, Assistant Professor of Bioengineering at University of Pennsylvania</li><br />
<li><b>Dan Cohen</b>, PhD, Postdoctoral Fellow at University of Pennsylvania</li><br />
<li><b>Henry Ma</b>, Engineer at University of Pennsylvania Bioengineering Instructional Laboratories</li><br />
<li><b>Karsticum Computing Inc.</b>, Software development company that was instrumental in the construction of VerifiGEM</li></ul><br />
</div><br />
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</html></div>Qiaophttp://2012.igem.org/Team:Penn/PartsTeam:Penn/Parts2012-10-27T03:06:36Z<p>Qiaop: </p>
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<b><div class="name" align="center">Parts Submitted</div></b><br><br />
<b><div class="name">INPNC-MCS (Multiple Cloning Site)</div></b><br />
A multiple cloning site version of Ice Nucleation Protein that can be used for surface display experiments. The associated MCS and GS short amino acid linker allows for rapid generation of fusion proteins for surface display. Expression of INPNC fusion proteins will traffic to the cell membrane. See <a href="http://partsregistry.org/Part:BBa_K811005"> BBa_K811005 </a></p><br />
<br><br />
<b><div class="name">ClyA</div></b><br />
<p style="color:black"> Cytolysin A is a 34kDa protein that is capable of inducing rapid cell lysis in mammalian cells through the formation of a pore complex in the plasma membrane. See <a href="http://partsregistry.org/Part:BBa_K811000">BBa_K811000</a> </p><br />
<br><br />
<b><div class="name">His-ClyA</div></b><br />
<p style="color:black"> A N-terminus his-tagged version of clyA that is ideal for IMAC protein purification and western blotting. See <a href="http://partsregistry.org/Part:BBa_K811001"> BBa_K811001 </a></p><br />
<br><br />
<b><div class="name">ClyA-His</div></b><br />
<p style="color:black"> A C-terminus his-tagged version of clyA that is ideal for IMAC protein purification and western blotting. See <a href="http://partsregistry.org/Part:BBa_K811002"> BBa_K811002 </a></p><br />
<br><br />
<b><div class="name">INPNC</div></b><br />
<p style="color:black"> A truncated N-C terminus version of Ice Nucleation Protein that can be used for surface display experiments. Expression of INPNC fusion proteins will traffic to the cell membrane. See <a href="http://partsregistry.org/Part:BBa_K811003"> BBa_K811003 </a></p><br />
<br><br />
<b><div class="name">INPNC-HA</div></b><br />
<p style="color:black"> A C-terminus HA-tagged version of Ice Nucleation Protein that can be used for surface display experiments. The HA tag allows for visualization of INPNC localization through immunocytochemistry and western blotting. Expression of INPNC fusion proteins will traffic to the cell membrane. See <a href="http://partsregistry.org/Part:BBa_K811004"> BBa_K811004 </a></p><br />
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<b><div class="name" align="center">Overview of Part BBa_K811005: INPNC-MCS<br />
</div></b><br /><br />
<p style="color:black;text-indent:30px;"><br />
Ice nucleation protein (INP) is a protein found in <i>Xanthomonas campestris</i> pc. campestris BCRC 12846. Its function is to provide a surface for ice nucleation, which results in the formation of ice crystals. However, recent studies have utilized INP for its surface display properties. In nature, the protein is anchored in the membrane through a glycosylphosphatidylinositol (GPI) anchor, a relatively rare occurrence in prokaryotes.</p><br />
</div><br />
<br />
<br />
<br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Experience<br />
</div></b><br /><br />
<p style="color:black;text-indent:30px;"><br />
Part BBa_K811005 has been utilized by the 2012 Penn iGEM team to display a variety of different proteins. The red fluorescent protein mCherry has been successfully displayed on the surface of E. Coli, where it can produce fluorescence. We displayed mCherry on the outer membrane of E. coli BL21. After sonication and centrifugation of induced cells, almost all of the mCherry was localized in the membrane fraction when fused to INPNC, whereas in the control Intein-mCherry fusion (which exhibits cytoplasmic localization), all of the mCherry was contained in the lysate (Figure 1).</p><br />
<br />
<div class="fig"><div align="center"><img src="https://static.igem.org/mediawiki/2012/a/a2/MCherry-vs-INPNC-mCherry.jpg" width="250" height="350"/><br><br />
<b>Figure 1</b></div>Figure 1: Surface display of mCherry using INPNC system. INPNC-mCherry and Intein-mCherry fusions were expressed in E. coli BL21 in the pET26b expression vector and Wood-Intein expression plasmid, respectively. When fused to INPNC, almost all mCherry was localized in the membrane fraction after sonication and centrifugation, while in the case of Intein-mCherry, all mCherry was localized in the cytoplasmic lysate.</div><br />
<br />
<br />
Furthermore, a HER2 binding protein, DARPin H20-2-G3 has also displayed on the surface of E. Coli, and has been shown to retain its HER2 binding affinity upon surface display through Part BBa_K811005.<br />
</div><br />
<br />
<br />
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<br><br />
<br />
<br />
<br />
<br />
<br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Overview<br />
</div></b><br /><br />
<p style="color:black;text-indent:30px;"><br />
Ice nucleation protein (INP) is a protein found in <i>Xanthomonas campestris</i> pc. campestris BCRC 12846. Its function is to provide a surface for ice nucleation, which results in the formation of ice crystals. However, recent studies have utilized INP for its surface display properties. In nature, the protein is anchored in the membrane through a glycosylphosphatidylinositol (GPI) anchor, a relatively rare occurrence in prokaryotes.</p><br />
</div><br />
<br />
<br />
<br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Experience<br />
</div></b><br /><br />
<p style="color:black;text-indent:30px;"><br />
Part BBa_K811005 has been utilized by the 2012 Penn iGEM team to display a variety of different proteins. The red fluorescent protein mCherry has been successfully displayed on the surface of E. Coli, where it can produce fluorescence. We displayed mCherry on the outer membrane of E. coli BL21. After sonication and centrifugation of induced cells, almost all of the mCherry was localized in the membrane fraction when fused to INPNC, whereas in the control Intein-mCherry fusion (which exhibits cytoplasmic localization), all of the mCherry was contained in the lysate (Figure 1).</p><br />
<br />
<div class="fig"><div align="center"><img src="https://static.igem.org/mediawiki/2012/a/a2/MCherry-vs-INPNC-mCherry.jpg" width="250" height="350"/><br><br />
<b>Figure 1</b></div>Figure 1: Surface display of mCherry using INPNC system. INPNC-mCherry and Intein-mCherry fusions were expressed in E. coli BL21 in the pET26b expression vector and Wood-Intein expression plasmid, respectively. When fused to INPNC, almost all mCherry was localized in the membrane fraction after sonication and centrifugation, while in the case of Intein-mCherry, all mCherry was localized in the cytoplasmic lysate.</div><br />
<br />
<br />
Furthermore, a HER2 binding protein, DARPin H20-2-G3 has also displayed on the surface of E. Coli, and has been shown to retain its HER2 binding affinity upon surface display through Part BBa_K811005.<br />
</div><br />
<br />
<br />
</html></div>Qiaophttp://2012.igem.org/Team:Penn/SurfaceDisplayBBaTeam:Penn/SurfaceDisplayBBa2012-10-27T02:44:56Z<p>Qiaop: </p>
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<br><br />
<br />
<br />
<br />
<br />
<br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Overview<br />
</div></b><br /><br />
<p style="color:black;text-indent:30px;"><br />
Ice nucleation protein (INP) is a protein found in <i>Xanthomonas campestris</i> pc. campestris BCRC 12846. Its function is to provide a surface for ice nucleation, which results in the formation of ice crystals. However, recent studies have utilized INP for its surface display properties. In nature, the protein is anchored in the membrane through a glycosylphosphatidylinositol (GPI) anchor, a relatively rare occurrence in prokaryotes.</p><br />
</div><br />
<br />
<br />
<br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Experience<br />
</div></b><br /><br />
<p style="color:black;text-indent:30px;"><br />
Part BBa_K811005 has been utilized by the 2012 Penn iGEM team to display a variety of different proteins. The red fluorescent protein mCherry has been successfully displayed on the surface of E. Coli, where it can produce fluorescence. We displayed mCherry on the outer membrane of E. coli BL21. After sonication and centrifugation of induced cells, almost all of the mCherry was localized in the membrane fraction when fused to INPNC, whereas in the control Intein-mCherry fusion (which exhibits cytoplasmic localization), all of the mCherry was contained in the lysate (Figure 1).</p><br />
<br />
<div class="fig"><div align="center"><img src="https://static.igem.org/mediawiki/2012/a/a2/MCherry-vs-INPNC-mCherry.jpg" width="250" height="350"/><br><br />
<b>Figure 1</b></div>Figure 1: Surface display of mCherry using INPNC system. INPNC-mCherry and Intein-mCherry fusions were expressed in E. coli BL21 in the pET26b expression vector and Wood-Intein expression plasmid, respectively. When fused to INPNC, almost all mCherry was localized in the membrane fraction after sonication and centrifugation, while in the case of Intein-mCherry, all mCherry was localized in the cytoplasmic lysate.</div><br />
<br />
<br />
Furthermore, a HER2 binding protein, DARPin H20-2-G3 has also displayed on the surface of E. Coli, and has been shown to retain its HER2 binding affinity upon surface display through Part BBa_K811005.<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Generalized Surface Display System<br />
</div></b><br /><br />
<p style="color:black;text-indent:30px;"><br />
We sought to create a system in which iGEM teams and labs can display any protein of their choosing on the surface of E. coli. We engineered a novel Suface Display BioBrick (BBa K811004) using the N and C terminal domains of the Ice Nucleation Protein (INPNC), which allows iGEM teams to fuse any desired protein to INPNC using a simple ligation protocol with BamHI and PstI restriction sites.</p><br />
<br />
<p style="color:black;text-indent:30px;"><br />
As a preliminary proof of concept for our INPNC-enabled system, we displayed the red fluorescent protein mCherry on the outer membrane of E. coli BL21. After sonication and centrifugation of induced cells, almost all of the mCherry was localized in the membrane fraction when fused to INPNC, whereas in the control Intein-mCherry fusion (which exhibits cytoplasmic localization), all of the mCherry was contained in the lysate (Figure 1).</p><br />
<br />
<br />
<div class="fig"><div align="center"><img src="https://static.igem.org/mediawiki/2012/a/a2/MCherry-vs-INPNC-mCherry.jpg" width="250" height="350"/><br><br />
<b>Figure 1</b></div>Figure 1: Surface display of mCherry using INPNC system. INPNC-mCherry and Intein-mCherry fusions were expressed in E. coli BL21 in the pET26b expression vector and Wood-Intein expression plasmid, respectively. When fused to INPNC, almost all mCherry was localized in the membrane fraction after sonication and centrifugation, while in the case of Intein-mCherry, all mCherry was localized in the cytoplasmic lysate.</div><br />
</div><br />
</html></div>Qiaophttp://2012.igem.org/Team:Penn/NissleTeam:Penn/Nissle2012-10-27T02:37:42Z<p>Qiaop: </p>
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<div class="bigbox"><br />
<b><div class="name" align="center">Use of the Minimally Immunogenic E. Coli Strain Nissle 1917</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"> We have seen how the complex interplay between public opinion and science innovation can drastically affect the adoption and success of a new technology, such as our team's bacterial drug delivery system. As pointed out earlier, pervading public opinion towards a bacterial therapeutic system such as the one developed by our team this year would most likely be negative. In an effort to address some of these issues, we have set out to investigate ways our system could be made more palatable to the general public. </p><br />
<p style="color:black;text-indent:30px;"> One recent concept that we have identified as gaining general acceptance within the general public is the incorporation of "probiotic" organisms into a daily diet. Many foods, such as yogurts now advertise the presence of "probiotic" bacteria and there are "probiotic" supplements containing live bacterial cultures as well. One particular probiotic, E. Coli Nissle 1917 has attracted attention not only from the public, but also from the scientific community, where its potential beneficial properties have been investigated. The Nissle strain is notable for its lack of virulence factors and decreased immunogenecity [1]. These traits are what make Nissle a popular probiotic. Nissle has also been found to preferentially colonize tumors, proliferating wildly in the borders between live and necrotic tissue, a highly desirable trait for any potential cancer treatment [2]. Additional investigation has demonstrated that intravenously administered Nissle exhibits a similar behavior in breast cancer mouse models, and expression of recombinant azurin prevented cancer metastasis in mice [4]. However, the therapeutic potential of Nissle is not limited to cancer treatment. Nissle is also capable of enhancing wound healing through recombinant expression of human epidermal Growth Factor in the epithelial linings of the body, as well as reducing modulating responses to allergens [5,6].</p><br />
<p style="color:black;text-indent:30px;"> Based on these properties, we believe that demonstrating that our drug delivery system can be implemented in Nissle 1917 would be the first step to addressing the potential hesitance that the public may have to bacterial based therapies. Because the chassis for our system is a probiotic, we can avoid not only the technical difficulties of ensuring that the host for our system is inherently safe, but also proactively address (or at least minimize) the initial "knee-jerk" reactions that many members of the general public may have to the idea of a bacterial therapeutic. Furthermore, In order to fully realize the potential of Nissle, it is important to be able to easily and consistently manipulate and change its genetic information. Therefore, we have produced and characterized a process for generating chemically competent Nissle 1917 that can be produced in any standard microbiology lab, allowing future iGEM teams to unlock Nissle 1917's full potential.<br />
</div><br />
<div class="bigbox"><br />
<b><div class="name" align="center">Data</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
As shown below, chemically competent Nissle 1917 bacteria produced by the lab was capable of taking up the pDawn-ClyA light-based cytolysis module of our system. Furthermore, these bacteria can begin to express genes encoded on the plasmid, such as the kanR gene, which confers resistance to the Kanamycin found in the LB plates.</p><br />
</p><div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/9/90/20121004033558!IMG_3309.JPG" width="600" height="400"></div><br />
<br />
<p style="color:black;text-indent:30px;"><br />
As shown below, chemically competent Nissle 1917 bacteria produced by the lab was capable of taking up the pDawn-mCherry plasmid modeled on the pDawn-ClyA light-based cytolysis module of our system. Furthermore, these bacteria exhibit blue light-dependent expression of mCherry, a red fluorescent protein, as seen on the right of the photo.</p><br />
</p><div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/7/72/Nissle-1917-pDawn-mCherry-10-1-2012.jpg/400px-Nissle-1917-pDawn-mCherry-10-1-2012.jpg" width="240" height="360"></div><br />
</div><br />
<br />
<br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Production of Chemically Competent Nissle 1917 Cells</div></b><br />
<ol><br />
<li>Inoculate one colony from LB plate into 2 ml LB liquid medium. Shake at 37 °C<br />
overnight.</li><br />
<li>Inoculate 1-ml overnight cell culture into 100 ml LB medium (in a 500 ml flask).</li><br />
<li>Shake vigorously at 37 °C to OD600 ~0.25-0.3.</li><br />
<li>Chill the culture on ice for 15 min. Also make sure the 0.1M CaCl2<br />
solution and 0.1M CaCl2 plus 15% glycerol are on ice.</li><br />
<li>Centrifuge the cells for 10 min at 5000 g at 4°C.</li><br />
<li>Discard the medium and resuspend the cell pellet in 30-40 ml cold 0.1M CaCl2. Keep the cells on ice for 30 min.</li><br />
<li>Centrifuge the cells as above.</li><br />
<li>Remove the supernatant, and resuspend the cell pellet in 6 ml 0.1 M CaCl2<br />
solution plus 15% glycerol.</li><br />
<li>Pipet 0.4-0.5 ml of the cell suspension into sterile 1.5 ml micro-centrifuge tubes. Flash freeze these tubes in liquid nitrogen and then transfer them to the -80 C freezer.<br />
<ul><br />
<li><br />
Note: Successful transformations have occured with 100uL of cells + 1ug of DNA, however the efficency of cells made through this process is lower than that of Subcloning Efficency DH5a from Invitrogen. After flash freezing, competency of cells prepared through this protocol increases over time with additional storage time in -80°C for approximately 3 days.<br />
</li><br />
</ul><br />
</ol><br />
</div><br />
<div class="bigbox"><br />
<b><div class="name" align="center">References:</div></b><br><br />
<br />
[1] Grozdanov, L., U. Zahringer, G. Blum-Oehler, L. Brade, A. Henne, Y. A. Knirel, U.Schombel, J. Schulze, U. Sonnenborn, G. Gottschalk, J. Hacker, E. T. Rietschel, and U. Dobrindt. "A Single Nucleotide Exchange in the Wzy Gene Is Responsible for the Semirough O6 Lipopolysaccharide Phenotype and Serum Sensitivity of Escherichia Coli Strain Nissle 1917." Journal of Bacteriology 184.21 (2002): 5912-925. Print.<br><br />
<br><br />
[2] Stritzker, J., S. Weibel, P. Hill, T. Oelschlaeger, W. Goebel, and A. Szalay.<br />
<br />
"Tumor-specific Colonization, Tissue Distribution, and Gene Induction by Probiotic Escherichia Coli Nissle 1917 in Live Mice." International Journal of Medical Microbiology 297.3 (2007): 151-62. 19 Apr. 2007. Web. 29 Sept. 2012.<br><br />
<br><br />
[3] Weise, Christin, Yan Zhu, Dennis Ernst, Anja A. Kühl, and Margitta Worm. "Oral<br />
<br />
Administration of Escherichia Coli Nissle 1917 Prevents Allergen-induced Dermatitis in Mice." Experimental Dermatology 20.10 (2011): 805-09. 11 July 2011. Web. 29 Sept. 2012. <br><br />
<br><br />
[4] Zhang, Y., L. Xia, X. Zhang, X. Ding, F. Yan, and F. Wu. "Escherichia Coli Nissle 1917 Targets and Restrains Mouse B16 Melanoma and 4T1 Breast Tumor through the Expression of Azurin Protein." Applied Environmental Microbiology (n.d.): n. pag. 24 Aug. 2012. Web. 29 Sept. 2012.</div><br />
</html></div>Qiaophttp://2012.igem.org/Team:Penn/NissleTeam:Penn/Nissle2012-10-27T02:29:16Z<p>Qiaop: </p>
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.name{ font-size:20px;}<br />
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<br><br />
<div class="bigbox"><br />
<b><div class="name" align="center">Use of the Minimally Immunogenic E. Coli Strain Nissle 1917</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"> We have seen how the complex interplay between public opinion and science innovation can drastically affect the adoption and success of a new technology, such as our team's bacterial drug delivery system. As pointed out earlier, pervading public opinion towards a bacterial therapeutic system such as the one developed by our team this year would most likely be negative. In an effort to address some of these issues, we have set out to investigate ways our system could be made more palatable to the general public. </p><br />
<p style="color:black;text-indent:30px;"> One recent concept that we have identified as gaining general acceptance within the general public is the incorporation of "probiotic" organisms into a daily diet. Many foods, such as yogurts now advertise the presence of "probiotic" bacteria and there are "probiotic" supplements containing live bacterial cultures as well. One particular probiotic, E. Coli Nissle 1917 has attracted attention not only from the public, but also from the scientific community, where its potential beneficial properties have been investigated. The Nissle strain is notable for its lack of virulence factors and decreased immunogenecity [1]. These traits are what make Nissle a popular probiotic. Nissle has also been found to preferentially colonize tumors, proliferating wildly in the borders between live and necrotic tissue, a highly desirable trait for any potential cancer treatment [2]. Additional investigation has demonstrated that intravenously administered Nissle exhibits a similar behavior in breast cancer mouse models, and expression of recombinant azurin prevented cancer metastasis in mice [4]. However, the therapeutic potential of Nissle is not limited to cancer treatment. Nissle is also capable of enhancing wound healing through recombinant expression of human epidermal Growth Factor in the epithelial linings of the body, as well as reducing modulating responses to allergens [5,6].</p><br />
<p style="color:black;text-indent:30px;"> Based on these properties, we believe that demonstrating that our drug delivery system can be implemented in Nissle 1917 would be the first step to addressing the potential hesitance that the public may have to bacterial based therapies. Because the chassis for our system is a probiotic, we can avoid not only the technical difficulties of ensuring that the host for our system is inherently safe, but also proactively address (or at least minimize) the initial "knee-jerk" reactions that many members of the general public may have to the idea of a bacterial therapeutic. Furthermore, In order to fully realize the potential of Nissle, it is important to be able to easily and consistently manipulate and change its genetic information. Therefore, we have produced and characterized a process for generating chemically competent Nissle 1917 that can be produced in any standard microbiology lab, allowing future iGEM teams to unlock Nissle 1917's full potential.<br />
<br />
</p><div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/9/90/20121004033558!IMG_3309.JPG" width="600" height="400"></div><br />
<br />
<br />
</p><div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/7/72/Nissle-1917-pDawn-mCherry-10-1-2012.jpg/400px-Nissle-1917-pDawn-mCherry-10-1-2012.jpg" width="240" height="360"></div><br />
</div><br />
<br />
<br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Production of Chemically Competent Nissle 1917 Cells</div></b><br />
<ol><br />
<li>Inoculate one colony from LB plate into 2 ml LB liquid medium. Shake at 37 °C<br />
overnight.</li><br />
<li>Inoculate 1-ml overnight cell culture into 100 ml LB medium (in a 500 ml flask).</li><br />
<li>Shake vigorously at 37 °C to OD600 ~0.25-0.3.</li><br />
<li>Chill the culture on ice for 15 min. Also make sure the 0.1M CaCl2<br />
solution and 0.1M CaCl2 plus 15% glycerol are on ice.</li><br />
<li>Centrifuge the cells for 10 min at 5000 g at 4°C.</li><br />
<li>Discard the medium and resuspend the cell pellet in 30-40 ml cold 0.1M CaCl2. Keep the cells on ice for 30 min.</li><br />
<li>Centrifuge the cells as above.</li><br />
<li>Remove the supernatant, and resuspend the cell pellet in 6 ml 0.1 M CaCl2<br />
solution plus 15% glycerol.</li><br />
<li>Pipet 0.4-0.5 ml of the cell suspension into sterile 1.5 ml micro-centrifuge tubes. Flash freeze these tubes in liquid nitrogen and then transfer them to the -80 C freezer.<br />
<ul><br />
<li><br />
Note: Successful transformations have occured with 100uL of cells + 1ug of DNA, however the efficency of cells made through this process is lower than that of Subcloning Efficency DH5a from Invitrogen. After flash freezing, competency of cells prepared through this protocol increases over time with additional storage time in -80°C for approximately 3 days.<br />
</li><br />
</ul><br />
</ol><br />
</div><br />
<div class="bigbox"><br />
<b><div class="name" align="center">References:</div></b><br><br />
<br />
[1] Grozdanov, L., U. Zahringer, G. Blum-Oehler, L. Brade, A. Henne, Y. A. Knirel, U.Schombel, J. Schulze, U. Sonnenborn, G. Gottschalk, J. Hacker, E. T. Rietschel, and U. Dobrindt. "A Single Nucleotide Exchange in the Wzy Gene Is Responsible for the Semirough O6 Lipopolysaccharide Phenotype and Serum Sensitivity of Escherichia Coli Strain Nissle 1917." Journal of Bacteriology 184.21 (2002): 5912-925. Print.<br><br />
<br><br />
[2] Stritzker, J., S. Weibel, P. Hill, T. Oelschlaeger, W. Goebel, and A. Szalay.<br />
<br />
"Tumor-specific Colonization, Tissue Distribution, and Gene Induction by Probiotic Escherichia Coli Nissle 1917 in Live Mice." International Journal of Medical Microbiology 297.3 (2007): 151-62. 19 Apr. 2007. Web. 29 Sept. 2012.<br><br />
<br><br />
[3] Weise, Christin, Yan Zhu, Dennis Ernst, Anja A. Kühl, and Margitta Worm. "Oral<br />
<br />
Administration of Escherichia Coli Nissle 1917 Prevents Allergen-induced Dermatitis in Mice." Experimental Dermatology 20.10 (2011): 805-09. 11 July 2011. Web. 29 Sept. 2012. <br><br />
<br><br />
[4] Zhang, Y., L. Xia, X. Zhang, X. Ding, F. Yan, and F. Wu. "Escherichia Coli Nissle 1917 Targets and Restrains Mouse B16 Melanoma and 4T1 Breast Tumor through the Expression of Azurin Protein." Applied Environmental Microbiology (n.d.): n. pag. 24 Aug. 2012. Web. 29 Sept. 2012.</div><br />
</html></div>Qiaophttp://2012.igem.org/Team:Penn/NissleTeam:Penn/Nissle2012-10-27T02:28:52Z<p>Qiaop: </p>
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<b><div class="name" align="center">Use of the Minimally Immunogenic E. Coli Strain Nissle 1917</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"> We have seen how the complex interplay between public opinion and science innovation can drastically affect the adoption and success of a new technology, such as our team's bacterial drug delivery system. As pointed out earlier, pervading public opinion towards a bacterial therapeutic system such as the one developed by our team this year would most likely be negative. In an effort to address some of these issues, we have set out to investigate ways our system could be made more palatable to the general public. </p><br />
<p style="color:black;text-indent:30px;"> One recent concept that we have identified as gaining general acceptance within the general public is the incorporation of "probiotic" organisms into a daily diet. Many foods, such as yogurts now advertise the presence of "probiotic" bacteria and there are "probiotic" supplements containing live bacterial cultures as well. One particular probiotic, E. Coli Nissle 1917 has attracted attention not only from the public, but also from the scientific community, where its potential beneficial properties have been investigated. The Nissle strain is notable for its lack of virulence factors and decreased immunogenecity [1]. These traits are what make Nissle a popular probiotic. Nissle has also been found to preferentially colonize tumors, proliferating wildly in the borders between live and necrotic tissue, a highly desirable trait for any potential cancer treatment [2]. Additional investigation has demonstrated that intravenously administered Nissle exhibits a similar behavior in breast cancer mouse models, and expression of recombinant azurin prevented cancer metastasis in mice [4]. However, the therapeutic potential of Nissle is not limited to cancer treatment. Nissle is also capable of enhancing wound healing through recombinant expression of human epidermal Growth Factor in the epithelial linings of the body, as well as reducing modulating responses to allergens [5,6].</p><br />
<p style="color:black;text-indent:30px;"> Based on these properties, we believe that demonstrating that our drug delivery system can be implemented in Nissle 1917 would be the first step to addressing the potential hesitance that the public may have to bacterial based therapies. Because the chassis for our system is a probiotic, we can avoid not only the technical difficulties of ensuring that the host for our system is inherently safe, but also proactively address (or at least minimize) the initial "knee-jerk" reactions that many members of the general public may have to the idea of a bacterial therapeutic. Furthermore, In order to fully realize the potential of Nissle, it is important to be able to easily and consistently manipulate and change its genetic information. Therefore, we have produced and characterized a process for generating chemically competent Nissle 1917 that can be produced in any standard microbiology lab, allowing future iGEM teams to unlock Nissle 1917's full potential.<br />
<br />
</p><div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/9/90/20121004033558!IMG_3309.JPG" width="600" height="400"></div><br />
<br />
<br />
</p><div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/7/72/Nissle-1917-pDawn-mCherry-10-1-2012.jpg/400px-Nissle-1917-pDawn-mCherry-10-1-2012.jpg" width="240" height="360"></div><br />
</div><br />
</div><br />
<br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Production of Chemically Competent Nissle 1917 Cells</div></b><br />
<ol><br />
<li>Inoculate one colony from LB plate into 2 ml LB liquid medium. Shake at 37 °C<br />
overnight.</li><br />
<li>Inoculate 1-ml overnight cell culture into 100 ml LB medium (in a 500 ml flask).</li><br />
<li>Shake vigorously at 37 °C to OD600 ~0.25-0.3.</li><br />
<li>Chill the culture on ice for 15 min. Also make sure the 0.1M CaCl2<br />
solution and 0.1M CaCl2 plus 15% glycerol are on ice.</li><br />
<li>Centrifuge the cells for 10 min at 5000 g at 4°C.</li><br />
<li>Discard the medium and resuspend the cell pellet in 30-40 ml cold 0.1M CaCl2. Keep the cells on ice for 30 min.</li><br />
<li>Centrifuge the cells as above.</li><br />
<li>Remove the supernatant, and resuspend the cell pellet in 6 ml 0.1 M CaCl2<br />
solution plus 15% glycerol.</li><br />
<li>Pipet 0.4-0.5 ml of the cell suspension into sterile 1.5 ml micro-centrifuge tubes. Flash freeze these tubes in liquid nitrogen and then transfer them to the -80 C freezer.<br />
<ul><br />
<li><br />
Note: Successful transformations have occured with 100uL of cells + 1ug of DNA, however the efficency of cells made through this process is lower than that of Subcloning Efficency DH5a from Invitrogen. After flash freezing, competency of cells prepared through this protocol increases over time with additional storage time in -80°C for approximately 3 days.<br />
</li><br />
</ul><br />
</ol><br />
</div><br />
<div class="bigbox"><br />
<b><div class="name" align="center">References:</div></b><br><br />
<br />
[1] Grozdanov, L., U. Zahringer, G. Blum-Oehler, L. Brade, A. Henne, Y. A. Knirel, U.Schombel, J. Schulze, U. Sonnenborn, G. Gottschalk, J. Hacker, E. T. Rietschel, and U. Dobrindt. "A Single Nucleotide Exchange in the Wzy Gene Is Responsible for the Semirough O6 Lipopolysaccharide Phenotype and Serum Sensitivity of Escherichia Coli Strain Nissle 1917." Journal of Bacteriology 184.21 (2002): 5912-925. Print.<br><br />
<br><br />
[2] Stritzker, J., S. Weibel, P. Hill, T. Oelschlaeger, W. Goebel, and A. Szalay.<br />
<br />
"Tumor-specific Colonization, Tissue Distribution, and Gene Induction by Probiotic Escherichia Coli Nissle 1917 in Live Mice." International Journal of Medical Microbiology 297.3 (2007): 151-62. 19 Apr. 2007. Web. 29 Sept. 2012.<br><br />
<br><br />
[3] Weise, Christin, Yan Zhu, Dennis Ernst, Anja A. Kühl, and Margitta Worm. "Oral<br />
<br />
Administration of Escherichia Coli Nissle 1917 Prevents Allergen-induced Dermatitis in Mice." Experimental Dermatology 20.10 (2011): 805-09. 11 July 2011. Web. 29 Sept. 2012. <br><br />
<br><br />
[4] Zhang, Y., L. Xia, X. Zhang, X. Ding, F. Yan, and F. Wu. "Escherichia Coli Nissle 1917 Targets and Restrains Mouse B16 Melanoma and 4T1 Breast Tumor through the Expression of Azurin Protein." Applied Environmental Microbiology (n.d.): n. pag. 24 Aug. 2012. Web. 29 Sept. 2012.</div><br />
</html></div>Qiaophttp://2012.igem.org/Team:Penn/NissleTeam:Penn/Nissle2012-10-27T02:27:20Z<p>Qiaop: </p>
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<b><div class="name" align="center">Use of the Minimally Immunogenic E. Coli Strain Nissle 1917</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"> We have seen how the complex interplay between public opinion and science innovation can drastically affect the adoption and success of a new technology, such as our team's bacterial drug delivery system. As pointed out earlier, pervading public opinion towards a bacterial therapeutic system such as the one developed by our team this year would most likely be negative. In an effort to address some of these issues, we have set out to investigate ways our system could be made more palatable to the general public. </p><br />
<p style="color:black;text-indent:30px;"> One recent concept that we have identified as gaining general acceptance within the general public is the incorporation of "probiotic" organisms into a daily diet. Many foods, such as yogurts now advertise the presence of "probiotic" bacteria and there are "probiotic" supplements containing live bacterial cultures as well. One particular probiotic, E. Coli Nissle 1917 has attracted attention not only from the public, but also from the scientific community, where its potential beneficial properties have been investigated. The Nissle strain is notable for its lack of virulence factors and decreased immunogenecity [1]. These traits are what make Nissle a popular probiotic. Nissle has also been found to preferentially colonize tumors, proliferating wildly in the borders between live and necrotic tissue, a highly desirable trait for any potential cancer treatment [2]. Additional investigation has demonstrated that intravenously administered Nissle exhibits a similar behavior in breast cancer mouse models, and expression of recombinant azurin prevented cancer metastasis in mice [4]. However, the therapeutic potential of Nissle is not limited to cancer treatment. Nissle is also capable of enhancing wound healing through recombinant expression of human epidermal Growth Factor in the epithelial linings of the body, as well as reducing modulating responses to allergens [5,6].</p><br />
<p style="color:black;text-indent:30px;"> Based on these properties, we believe that demonstrating that our drug delivery system can be implemented in Nissle 1917 would be the first step to addressing the potential hesitance that the public may have to bacterial based therapies. Because the chassis for our system is a probiotic, we can avoid not only the technical difficulties of ensuring that the host for our system is inherently safe, but also proactively address (or at least minimize) the initial "knee-jerk" reactions that many members of the general public may have to the idea of a bacterial therapeutic. Furthermore, In order to fully realize the potential of Nissle, it is important to be able to easily and consistently manipulate and change its genetic information. Therefore, we have produced and characterized a process for generating chemically competent Nissle 1917 that can be produced in any standard microbiology lab, allowing future iGEM teams to unlock Nissle 1917's full potential.<br />
<br />
</p><div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/9/90/20121004033558!IMG_3309.JPG" width="600" height="400"></div><br />
</div><br />
<br />
</p><div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/7/72/Nissle-1917-pDawn-mCherry-10-1-2012.jpg/400px-Nissle-1917-pDawn-mCherry-10-1-2012.jpg" width="240" height="360"></div><br />
</div><br />
<br />
<br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Production of Chemically Competent Nissle 1917 Cells</div></b><br />
<ol><br />
<li>Inoculate one colony from LB plate into 2 ml LB liquid medium. Shake at 37 °C<br />
overnight.</li><br />
<li>Inoculate 1-ml overnight cell culture into 100 ml LB medium (in a 500 ml flask).</li><br />
<li>Shake vigorously at 37 °C to OD600 ~0.25-0.3.</li><br />
<li>Chill the culture on ice for 15 min. Also make sure the 0.1M CaCl2<br />
solution and 0.1M CaCl2 plus 15% glycerol are on ice.</li><br />
<li>Centrifuge the cells for 10 min at 5000 g at 4°C.</li><br />
<li>Discard the medium and resuspend the cell pellet in 30-40 ml cold 0.1M CaCl2. Keep the cells on ice for 30 min.</li><br />
<li>Centrifuge the cells as above.</li><br />
<li>Remove the supernatant, and resuspend the cell pellet in 6 ml 0.1 M CaCl2<br />
solution plus 15% glycerol.</li><br />
<li>Pipet 0.4-0.5 ml of the cell suspension into sterile 1.5 ml micro-centrifuge tubes. Flash freeze these tubes in liquid nitrogen and then transfer them to the -80 C freezer.<br />
<ul><br />
<li><br />
Note: Successful transformations have occured with 100uL of cells + 1ug of DNA, however the efficency of cells made through this process is lower than that of Subcloning Efficency DH5a from Invitrogen. After flash freezing, competency of cells prepared through this protocol increases over time with additional storage time in -80°C for approximately 3 days.<br />
</li><br />
</ul><br />
</ol><br />
</div><br />
<div class="bigbox"><br />
<b><div class="name" align="center">References:</div></b><br><br />
<br />
[1] Grozdanov, L., U. Zahringer, G. Blum-Oehler, L. Brade, A. Henne, Y. A. Knirel, U.Schombel, J. Schulze, U. Sonnenborn, G. Gottschalk, J. Hacker, E. T. Rietschel, and U. Dobrindt. "A Single Nucleotide Exchange in the Wzy Gene Is Responsible for the Semirough O6 Lipopolysaccharide Phenotype and Serum Sensitivity of Escherichia Coli Strain Nissle 1917." Journal of Bacteriology 184.21 (2002): 5912-925. Print.<br><br />
<br><br />
[2] Stritzker, J., S. Weibel, P. Hill, T. Oelschlaeger, W. Goebel, and A. Szalay.<br />
<br />
"Tumor-specific Colonization, Tissue Distribution, and Gene Induction by Probiotic Escherichia Coli Nissle 1917 in Live Mice." International Journal of Medical Microbiology 297.3 (2007): 151-62. 19 Apr. 2007. Web. 29 Sept. 2012.<br><br />
<br><br />
[3] Weise, Christin, Yan Zhu, Dennis Ernst, Anja A. Kühl, and Margitta Worm. "Oral<br />
<br />
Administration of Escherichia Coli Nissle 1917 Prevents Allergen-induced Dermatitis in Mice." Experimental Dermatology 20.10 (2011): 805-09. 11 July 2011. Web. 29 Sept. 2012. <br><br />
<br><br />
[4] Zhang, Y., L. Xia, X. Zhang, X. Ding, F. Yan, and F. Wu. "Escherichia Coli Nissle 1917 Targets and Restrains Mouse B16 Melanoma and 4T1 Breast Tumor through the Expression of Azurin Protein." Applied Environmental Microbiology (n.d.): n. pag. 24 Aug. 2012. Web. 29 Sept. 2012.</div><br />
</html></div>Qiaophttp://2012.igem.org/Team:Penn/NissleTeam:Penn/Nissle2012-10-27T02:25:31Z<p>Qiaop: </p>
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<b><div class="name" align="center">Use of the Minimally Immunogenic E. Coli Strain Nissle 1917</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"> We have seen how the complex interplay between public opinion and science innovation can drastically affect the adoption and success of a new technology, such as our team's bacterial drug delivery system. As pointed out earlier, pervading public opinion towards a bacterial therapeutic system such as the one developed by our team this year would most likely be negative. In an effort to address some of these issues, we have set out to investigate ways our system could be made more palatable to the general public. </p><br />
<p style="color:black;text-indent:30px;"> One recent concept that we have identified as gaining general acceptance within the general public is the incorporation of "probiotic" organisms into a daily diet. Many foods, such as yogurts now advertise the presence of "probiotic" bacteria and there are "probiotic" supplements containing live bacterial cultures as well. One particular probiotic, E. Coli Nissle 1917 has attracted attention not only from the public, but also from the scientific community, where its potential beneficial properties have been investigated. The Nissle strain is notable for its lack of virulence factors and decreased immunogenecity [1]. These traits are what make Nissle a popular probiotic. Nissle has also been found to preferentially colonize tumors, proliferating wildly in the borders between live and necrotic tissue, a highly desirable trait for any potential cancer treatment [2]. Additional investigation has demonstrated that intravenously administered Nissle exhibits a similar behavior in breast cancer mouse models, and expression of recombinant azurin prevented cancer metastasis in mice [4]. However, the therapeutic potential of Nissle is not limited to cancer treatment. Nissle is also capable of enhancing wound healing through recombinant expression of human epidermal Growth Factor in the epithelial linings of the body, as well as reducing modulating responses to allergens [5,6].</p><br />
<p style="color:black;text-indent:30px;"> Based on these properties, we believe that demonstrating that our drug delivery system can be implemented in Nissle 1917 would be the first step to addressing the potential hesitance that the public may have to bacterial based therapies. Because the chassis for our system is a probiotic, we can avoid not only the technical difficulties of ensuring that the host for our system is inherently safe, but also proactively address (or at least minimize) the initial "knee-jerk" reactions that many members of the general public may have to the idea of a bacterial therapeutic. Furthermore, In order to fully realize the potential of Nissle, it is important to be able to easily and consistently manipulate and change its genetic information. Therefore, we have produced and characterized a process for generating chemically competent Nissle 1917 that can be produced in any standard microbiology lab, allowing future iGEM teams to unlock Nissle 1917's full potential.<br />
<br />
</p><div align="jusfify"><br />
<img src="https://static.igem.org/mediawiki/2012/9/90/20121004033558!IMG_3309.JPG" width="600" height="400"></div><br />
</div><br />
<br />
<br />
<br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Production of Chemically Competent Nissle 1917 Cells</div></b><br />
<ol><br />
<li>Inoculate one colony from LB plate into 2 ml LB liquid medium. Shake at 37 °C<br />
overnight.</li><br />
<li>Inoculate 1-ml overnight cell culture into 100 ml LB medium (in a 500 ml flask).</li><br />
<li>Shake vigorously at 37 °C to OD600 ~0.25-0.3.</li><br />
<li>Chill the culture on ice for 15 min. Also make sure the 0.1M CaCl2<br />
solution and 0.1M CaCl2 plus 15% glycerol are on ice.</li><br />
<li>Centrifuge the cells for 10 min at 5000 g at 4°C.</li><br />
<li>Discard the medium and resuspend the cell pellet in 30-40 ml cold 0.1M CaCl2. Keep the cells on ice for 30 min.</li><br />
<li>Centrifuge the cells as above.</li><br />
<li>Remove the supernatant, and resuspend the cell pellet in 6 ml 0.1 M CaCl2<br />
solution plus 15% glycerol.</li><br />
<li>Pipet 0.4-0.5 ml of the cell suspension into sterile 1.5 ml micro-centrifuge tubes. Flash freeze these tubes in liquid nitrogen and then transfer them to the -80 C freezer.<br />
<ul><br />
<li><br />
Note: Successful transformations have occured with 100uL of cells + 1ug of DNA, however the efficency of cells made through this process is lower than that of Subcloning Efficency DH5a from Invitrogen. After flash freezing, competency of cells prepared through this protocol increases over time with additional storage time in -80°C for approximately 3 days.<br />
</li><br />
</ul><br />
</ol><br />
</div><br />
<div class="bigbox"><br />
<b><div class="name" align="center">References:</div></b><br><br />
<br />
[1] Grozdanov, L., U. Zahringer, G. Blum-Oehler, L. Brade, A. Henne, Y. A. Knirel, U.Schombel, J. Schulze, U. Sonnenborn, G. Gottschalk, J. Hacker, E. T. Rietschel, and U. Dobrindt. "A Single Nucleotide Exchange in the Wzy Gene Is Responsible for the Semirough O6 Lipopolysaccharide Phenotype and Serum Sensitivity of Escherichia Coli Strain Nissle 1917." Journal of Bacteriology 184.21 (2002): 5912-925. Print.<br><br />
<br><br />
[2] Stritzker, J., S. Weibel, P. Hill, T. Oelschlaeger, W. Goebel, and A. Szalay.<br />
<br />
"Tumor-specific Colonization, Tissue Distribution, and Gene Induction by Probiotic Escherichia Coli Nissle 1917 in Live Mice." International Journal of Medical Microbiology 297.3 (2007): 151-62. 19 Apr. 2007. Web. 29 Sept. 2012.<br><br />
<br><br />
[3] Weise, Christin, Yan Zhu, Dennis Ernst, Anja A. Kühl, and Margitta Worm. "Oral<br />
<br />
Administration of Escherichia Coli Nissle 1917 Prevents Allergen-induced Dermatitis in Mice." Experimental Dermatology 20.10 (2011): 805-09. 11 July 2011. Web. 29 Sept. 2012. <br><br />
<br><br />
[4] Zhang, Y., L. Xia, X. Zhang, X. Ding, F. Yan, and F. Wu. "Escherichia Coli Nissle 1917 Targets and Restrains Mouse B16 Melanoma and 4T1 Breast Tumor through the Expression of Azurin Protein." Applied Environmental Microbiology (n.d.): n. pag. 24 Aug. 2012. Web. 29 Sept. 2012.</div><br />
</html></div>Qiaophttp://2012.igem.org/File:Nissle-1917-pDawn-mCherry-10-1-2012.jpgFile:Nissle-1917-pDawn-mCherry-10-1-2012.jpg2012-10-27T02:23:47Z<p>Qiaop: uploaded a new version of &quot;File:Nissle-1917-pDawn-mCherry-10-1-2012.jpg&quot;</p>
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<div></div>Qiaophttp://2012.igem.org/Team:Penn/BLSensorTeam:Penn/BLSensor2012-10-27T02:22:51Z<p>Qiaop: </p>
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<b><div class="name" align="center">YF1/FixJ (pDawn) Objectives </div></b><br />
<br><br />
To characterize our pDawn gene expression system, we showed the following:<br />
<ol><br />
<li> pDawn allows for light-dependent gene expression in bacteria<br />
<li> pDawn allows for light-dependent lysis of mammalian cells by bacteria<br />
</ol><br />
<br />
</div><br />
<br />
<br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Light Dependent Gene Expression in Bacteria</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;">We tested for light dependent gene expression by cloning in an mCherry reporter protein into the multiple cloning site of the pDawn system. First, we tested for the on-off ratio by growing cultures of BL21-pDawn-mCherry in both inducing and non-inducing conditions for 22 hours. After spinning down the cultures in a centrifuge, we were able to visually confirm the expression of mCherry due to the bacterial pellet grown in inducing conditions to be colored red, while the other pellet had no color (Figure 1).</p><br />
<br />
<div align="center"><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/f/f4/Clark_Park_4.JPG" width="180" height="300"><br />
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</div><br />
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<div style="text-align:center"><b>Figure 1</b><br /></div><br />
</div><br />
<br />
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<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Characterizing Time-Dependent Gene Expression</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
We then characterized the induction kinetics of the pDawn system through an mCherry expression time course. We induced cultures of BL21 pDawn-mCherry for 0 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, or 22 hours in a 37C incubator shaking at 225 rpm, and then transferred them into a dark incubator under the same condition for the remaining growth period. After 24 hours, mCherry fluorescence was read on a Tecan Infinite m200 plate reader and normalized by OD. The cultures were then spun down in a centrifuge. These results can be seen in Figure 2. </p><br />
<br><br />
<div align="center"><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/2/2a/PDawn-mCherry-Timecourse.gif" width="500"><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/b/b9/Timecourse.png" width="500" ><br />
<br />
<br />
</div><br />
<br><br />
<div style="text-align:center"><b>Figure 2</b><br /></div><br />
</div><br />
<div class="bigbox"><br />
<b><div class="name" align="center">pDawn and Nissle 1917</div></b><br /><br />
<br />
<p style="color:black;text-indent:30px;"><br />
In order to further develop our system for future in vivo therapeutic applications, we transformed Nissle 1917 with pDawn-mCherry to see if we could implement our system into a non-pathogenic strain of E. coli. We repeated our initial experiments and achieved light-dependent gene expression in Nissle 1917 for the first time ever. We are now hoping to clone in our pDawn-ClyA construct to show that Nissle 1917 is capable of light-dependent lysis of mammalian cells. Stay tuned!<br />
</p><br />
<br />
<div class="fig"><div align="center"><img src="https://static.igem.org/mediawiki/2012/7/72/Nissle-1917-pDawn-mCherry-10-1-2012.jpg" width="250" height="350"><br><br />
<br><br />
<b>Figure 3</b></div></div><br />
</html></div>Qiaophttp://2012.igem.org/Team:Penn/HumanPracticesOverviewTeam:Penn/HumanPracticesOverview2012-10-27T02:16:05Z<p>Qiaop: </p>
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.smallbox{ margin:20px auto; width:600px; padding:40px;background:#d7dce1;border:2px solid #708090;border-radius:10px;-moz-border-radius:10px;-webkit-border-radius:10px; overflow:hidden; text-align:justify;}<br />
.pic1{ float:left; margin:0 40px 0 0; width:150px;}<br />
.name{ font-size:20px;}<br />
</style><br />
<br><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Human Practices: Transitioning From the Bench to the Bedside</div></b><br />
<br><br />
<p style="text-align:justify;">Many previous iGEM teams have tried to implement a bacterial therapeutic as part of their project. Outside of iGEM, there has been a steady interest in engineering bacteria to become therapeutic vectors as well. However, the question that guided our human practices project was essentially: <b><br><p style="text-align:center">Why aren't bacterial therapeutics transitioning into clinical practice or even clinical trials? </p></p></b><br />
<p style="text-align:justify;"><br />
While there are certainly many barriers to bacterial therapeutics such as time and money, we hypothesize that iGEM teams, as a result of their unique positions as research and educational institutions, are positioned to address two major barriers to the adoption of bacterial therapeutics: biological barriers and perception barriers.<br />
</p><br />
<br><br />
</div><br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Biological Barriers to Bacterial Therapeutics</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
From a technological standpoint, there is a great deal of work that remains to be done before a bacterial therapeutic can enter the drug development pipeline. While many iGEM teams, including us, have helped set the groundwork for bacterial therapeutics, there are still some biological barriers to a bacterial therapeutic. We identified the immunogenicity of laboratory strains of <i>E. coli.</i> as a major biological barrier. We then investigated methods to decrease the immunogenicity of <i>E. coli.</i>, eventually choosing to port modules of our target drug delivery system into a non-immunogenic strain of <i>E. coli.</i>, Nissle 1917. <br />
</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/c/c6/VerifiGEM-Logo.jpg/800px-VerifiGEM-Logo.jpg" width="900" height="200" /></div><br />
<p style="color:black;text-indent:30px;"><br />
Furthermore, in an effort to speed progress and information sharing between iGEM teams, government research organizations, and private research groups, we have proposed a system known as VerifiGEM that would allow for the quality control of BioBricks to be distributed across the entire iGEM community. Should our system be implemented, the overall quality and reliability of BioBricks will be improved greatly, at very little cost to individual iGEM teams. <br />
</p><br />
</div><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Perception Barriers to Bacterial Therapeutics</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
However, the removal of biological barriers to bacterial therapeutics alone is not sufficient to enable bacterial therapeutics to move into the drug development pipeline. Through the course of recent history, many high profile technologies, such as gene therapy or nanotechnology have been met with public skepticism and even fear, as the technologies failed to deliver on earlier promises. This prompted a constriction of available funding and subsequently impeded progress in those fields, an outcome that nanotechnology in particular is only just beginning to recover from.<br />
</p><br />
<p style="color:black;text-indent:30px;"><br />
We propose a model, adapted from Gartner Inc, which proposes that the disconnect between the expectations of the public and the realities of scientific research produces an initial "peak of inflated expectations" (and funding), that rapidly disappears as promised advances are delayed or do not work as planned (Figure 1). We believe that this "trough of disillusionment" is the cause of restrictions in funding and a general stall of scientific progress in a given field. We propose that the peak can be smoothed out, reducing the size of peak, but more importantly, eliminating the trough of disillusionment (Figure 2).<br />
</p><br />
<br />
<div class="figs2"><br />
<div class="fignew"><div align="center"><img src="https://static.igem.org/mediawiki/2012/7/7c/Hype-Cycle-Original.jpg" height="200" width="320"><br><br />
<b>Figure 10</b></div>Figure 1: The hype cycle without the modulating effects of public outreach efforts.</div><br />
<br />
<div class="fignew"><div align="center"><img src="https://static.igem.org/mediawiki/2012/a/ab/Hype-Cycle-Original---Copy.jpg" height="200" width="320"><br><br />
<b>Figure 11</b></div>Figure 2: The modulation of the extremes of the hype cycle through public outreach efforts such as iGEM. </div></div><br />
<br />
<br />
<p style="color:black;text-indent:30px;"><br />
What then, must we as synthetic biologists do to prevent this fate from befalling our own field of study? Certainly, scientists must perform a balancing act between reporting the advances they have made and making realistic conclusions. iGEM teams in particular have a unique opportunity to impact this cycle. Each time a team teaches a class to younger students, presents their research, or even sets up a fun experiment at a local science event, they are given an opportunity to communicate the potential and the limitations of synthetic biology. This is an opportunity many researchers do not have, and should be treated as more than simply a time to take pictures and have fun (although those are certainly important parts of these events nevertheless). <br />
</p><br />
</div><br />
</body><br />
</html></div>Qiaophttp://2012.igem.org/Team:Penn/HumanPracticesOverviewTeam:Penn/HumanPracticesOverview2012-10-27T02:15:23Z<p>Qiaop: </p>
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.smallbox{ margin:20px auto; width:600px; padding:40px;background:#d7dce1;border:2px solid #708090;border-radius:10px;-moz-border-radius:10px;-webkit-border-radius:10px; overflow:hidden; text-align:justify;}<br />
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.name{ font-size:20px;}<br />
</style><br />
<br><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Human Practices: Transitioning From the Bench to the Bedside</div></b><br />
<br><br />
<p style="text-align:justify;">Many previous iGEM teams have tried to implement a bacterial therapeutic as part of their project. Outside of iGEM, there has been a steady interest in engineering bacteria to become therapeutic vectors as well. However, the question that guided our human practices project was essentially: <b><br><p style="text-align:center">Why aren't bacterial therapeutics transitioning into clinical practice or even clinical trials? </p></p></b><br />
<p style="text-align:justify;"><br />
While there are certainly many barriers to bacterial therapeutics such as time and money, we hypothesize that iGEM teams, as a result of their unique positions as research and educational institutions, are positioned to address two major barriers to the adoption of bacterial therapeutics: biological barriers and perception barriers.<br />
</p><br />
<br><br />
</div><br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Biological Barriers to Bacterial Therapeutics</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
From a technological standpoint, there is a great deal of work that remains to be done before a bacterial therapeutic can enter the drug development pipeline. While many iGEM teams, including us, have helped set the groundwork for bacterial therapeutics, there are still some biological barriers to a bacterial therapeutic. We identified the immunogenicity of laboratory strains of <i>E. coli.</i> as a major biological barrier. We then investigated methods to decrease the immunogenicity of <i>E. coli.</i>, eventually choosing to port modules of our target drug delivery system into a non-immunogenic strain of <i>E. coli.</i>, Nissle 1917. <br />
</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/c/c6/VerifiGEM-Logo.jpg/800px-VerifiGEM-Logo.jpg" width="900" height="200" /></div><br />
<p style="color:black;text-indent:30px;"><br />
Furthermore, in an effort to speed progress and information sharing between iGEM teams, government research organizations, and private research groups, we have proposed a system known as VerifiGEM that would allow for the quality control of BioBricks to be distributed across the entire iGEM community. Should our system be implemented, the overall quality and reliability of BioBricks will be improved greatly, at very little cost to individual iGEM teams. <br />
</p><br />
</div><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Perception Barriers to Bacterial Therapeutics</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
However, the removal of biological barriers to bacterial therapeutics alone is not sufficient to enable bacterial therapeutics to move into the drug development pipeline. Through the course of recent history, many high profile technologies, such as gene therapy or nanotechnology have been met with public skepticism and even fear, as the technologies failed to deliver on earlier promises. This prompted a constriction of available funding and subsequently impeded progress in those fields, an outcome that nanotechnology in particular is only just beginning to recover from.<br />
</p><br />
<p style="color:black;text-indent:30px;"><br />
We propose a model, adapted from Gartner Inc, which proposes that the disconnect between the expectations of the public and the realities of scientific research produces an initial "peak of inflated expectations" (and funding), that rapidly disappears as promised advances are delayed or do not work as planned (Figure 1). We believe that this "trough of disillusionment" is the cause of restrictions in funding and a general stall of scientific progress in a given field. We propose that the peak can be smoothed out, reducing the size of peak, but more importantly, eliminating the trough of disillusionment (Figure 2).<br />
</p><br />
<br />
<div class="figs2"><br />
<div class="fignew"><div align="center"><img src="https://static.igem.org/mediawiki/2012/7/7c/Hype-Cycle-Original.jpg" height="400" width="640"><br><br />
<b>Figure 10</b></div>Figure 1: The hype cycle without the modulating effects of public outreach efforts.</div><br />
<br />
<div class="fignew"><div align="center"><img src="https://static.igem.org/mediawiki/2012/a/ab/Hype-Cycle-Original---Copy.jpg" height="400" width="640"><br><br />
<b>Figure 11</b></div>Figure 2: The modulation of the extremes of the hype cycle through public outreach efforts such as iGEM. </div></div><br />
<br />
<br />
<p style="color:black;text-indent:30px;"><br />
What then, must we as synthetic biologists do to prevent this fate from befalling our own field of study? Certainly, scientists must perform a balancing act between reporting the advances they have made and making realistic conclusions. iGEM teams in particular have a unique opportunity to impact this cycle. Each time a team teaches a class to younger students, presents their research, or even sets up a fun experiment at a local science event, they are given an opportunity to communicate the potential and the limitations of synthetic biology. This is an opportunity many researchers do not have, and should be treated as more than simply a time to take pictures and have fun (although those are certainly important parts of these events nevertheless). <br />
</p><br />
</div><br />
</body><br />
</html></div>Qiaophttp://2012.igem.org/Team:Penn/HumanPracticesOverviewTeam:Penn/HumanPracticesOverview2012-10-27T02:04:52Z<p>Qiaop: </p>
<hr />
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<br><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Human Practices: Transitioning From the Bench to the Bedside</div></b><br />
<br><br />
<p style="text-align:justify;">Many previous iGEM teams have tried to implement a bacterial therapeutic as part of their project. Outside of iGEM, there has been a steady interest in engineering bacteria to become therapeutic vectors as well. However, the question that guided our human practices project was essentially: <b><br><p style="text-align:center">Why aren't bacterial therapeutics transitioning into clinical practice or even clinical trials? </p></p></b><br />
<p style="text-align:justify;"><br />
While there are certainly many barriers to bacterial therapeutics such as time and money, we hypothesize that iGEM teams, as a result of their unique positions as research and educational institutions, are positioned to address two major barriers to the adoption of bacterial therapeutics: biological barriers and perception barriers.<br />
</p><br />
<br><br />
</div><br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Biological Barriers to Bacterial Therapeutics</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
From a technological standpoint, there is a great deal of work that remains to be done before a bacterial therapeutic can enter the drug development pipeline. While many iGEM teams, including us, have helped set the groundwork for bacterial therapeutics, there are still some biological barriers to a bacterial therapeutic. We identified the immunogenicity of laboratory strains of <i>E. coli.</i> as a major biological barrier. We then investigated methods to decrease the immunogenicity of <i>E. coli.</i>, eventually choosing to port modules of our target drug delivery system into a non-immunogenic strain of <i>E. coli.</i>, Nissle 1917. <br />
</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/c/c6/VerifiGEM-Logo.jpg/800px-VerifiGEM-Logo.jpg" width="900" height="200" /></div><br />
<p style="color:black;text-indent:30px;"><br />
Furthermore, in an effort to speed progress and information sharing between iGEM teams, government research organizations, and private research groups, we have proposed a system known as VerifiGEM that would allow for the quality control of BioBricks to be distributed across the entire iGEM community. Should our system be implemented, the overall quality and reliability of BioBricks will be improved greatly, at very little cost to individual iGEM teams. <br />
</p><br />
</div><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Perception Barriers to Bacterial Therapeutics</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
However, the removal of biological barriers to bacterial therapeutics alone is not sufficient to enable bacterial therapeutics to move into the drug development pipeline. Through the course of recent history, many high profile technologies, such as gene therapy or nanotechnology have been met with public skepticism and even fear, as the technologies failed to deliver on earlier promises. This prompted a constriction of available funding and subsequently impeded progress in those fields, an outcome that nanotechnology in particular is only just beginning to recover from.<br />
</p><br />
<p style="color:black;text-indent:30px;"><br />
We propose a model, adapted from Gartner Inc, which proposes that the disconnect between the expectations of the public and the realities of scientific research produces an initial "peak of inflated expectations" (and funding), that rapidly disappears as promised advances are delayed or do not work as planned (Figure 1). We believe that this "trough of disillusionment" is the cause of restrictions in funding and a general stall of scientific progress in a given field. We propose that the peak can be smoothed out, reducing the size of peak, but more importantly, eliminating the trough of disillusionment (Figure 2).<br />
</p><br />
<br />
<div class="figs2"><br />
<div class="fignew"><div align="center"><img src="https://static.igem.org/mediawiki/2012/7/7c/Hype-Cycle-Original.jpg" height="400" width="640"><br><br />
<b>Figure 10</b></div>Figure 1: The hype cycle without the modulating effects of public outreach efforts.</div><br />
<br />
<div class="fignew"><div align="center"><img src="https://static.igem.org/mediawiki/2012/a/ab/Hype-Cycle-Original---Copy.jpg" height="400" width="640"><br><br />
<b>Figure 11</b></div>Figure 11: Images of breast cancer cells removed from human tissue reveal similar HER 2 expression to results obtained in Figure 10. </div></div><br />
<br />
<br />
<p style="color:black;text-indent:30px;"><br />
What then, must we as synthetic biologists do to prevent this fate from befalling our own field of study? Certainly, scientists must perform a balancing act between reporting the advances they have made and making realistic conclusions. iGEM teams in particular have a unique opportunity to <br />
</p><br />
</div><br />
<br />
<br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Human Practices</div></b><br />
<br><br />
<br />
<p style="color:black;text-indent:30px;">Upon conception of this project, we realized that although hundreds of academic research projects and iGEM projects have been conducted in the realm of Health and Medicine, almost no engineered bacterial therapeutics have been brought to the clinic. We analyzed the hurdles and road ahead for bacterial synthetic biology-enabled therapeutics, compiling a thorough report with specific actions which iGEM teams in Health/Medicine can take to make their therapies more clinically tractable. This project directly informed our wet lab work, causing us to port our therapeutic system into a non-pathogenic, probiotic bacterial strain which is already used in human therapies today.</p><br />
<br />
<br />
<p style="color:black;text-indent:30px;">We hope our targeted therapeutic platform will allow other scientists and iGEM teams to target any cells they choose. In the near term, we are planning to test our cancer cell targeting/killing bacterial system in a mouse model and make a real impact on cancer research and therapy.</p><br />
</div><br />
</body><br />
</html></div>Qiaophttp://2012.igem.org/File:Hype-Cycle-Original---Copy.jpgFile:Hype-Cycle-Original---Copy.jpg2012-10-27T02:02:48Z<p>Qiaop: </p>
<hr />
<div></div>Qiaophttp://2012.igem.org/File:Hype-Cycle-Original.jpgFile:Hype-Cycle-Original.jpg2012-10-27T02:02:34Z<p>Qiaop: </p>
<hr />
<div></div>Qiaophttp://2012.igem.org/Team:Penn/HumanPracticesOverviewTeam:Penn/HumanPracticesOverview2012-10-27T02:00:16Z<p>Qiaop: </p>
<hr />
<div>{{:Team:Penn/Template/Site}}<br />
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.all{ width:1000px; margin:0 auto;}<br />
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.bigbox{width:936px; padding:30px;background:#d7dce1;border:2px solid #708090;border-radius:10px;-moz-border-radius:10px;-webkit-border-radius:10px;color:black; font-size:14px; text-align:justify; margin:0 0 20px 0;}<br />
.smallbox{ margin:20px auto; width:600px; padding:40px;background:#d7dce1;border:2px solid #708090;border-radius:10px;-moz-border-radius:10px;-webkit-border-radius:10px; overflow:hidden; text-align:justify;}<br />
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<br><br />
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<div class="bigbox"><br />
<b><div class="name" align="center">Human Practices: Transitioning From the Bench to the Bedside</div></b><br />
<br><br />
<p style="text-align:justify;">Many previous iGEM teams have tried to implement a bacterial therapeutic as part of their project. Outside of iGEM, there has been a steady interest in engineering bacteria to become therapeutic vectors as well. However, the question that guided our human practices project was essentially: <b><br><p style="text-align:center">Why aren't bacterial therapeutics transitioning into clinical practice or even clinical trials? </p></p></b><br />
<p style="text-align:justify;"><br />
While there are certainly many barriers to bacterial therapeutics such as time and money, we hypothesize that iGEM teams, as a result of their unique positions as research and educational institutions, are positioned to address two major barriers to the adoption of bacterial therapeutics: biological barriers and perception barriers.<br />
</p><br />
<br><br />
</div><br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Biological Barriers to Bacterial Therapeutics</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
From a technological standpoint, there is a great deal of work that remains to be done before a bacterial therapeutic can enter the drug development pipeline. While many iGEM teams, including us, have helped set the groundwork for bacterial therapeutics, there are still some biological barriers to a bacterial therapeutic. We identified the immunogenicity of laboratory strains of <i>E. coli.</i> as a major biological barrier. We then investigated methods to decrease the immunogenicity of <i>E. coli.</i>, eventually choosing to port modules of our target drug delivery system into a non-immunogenic strain of <i>E. coli.</i>, Nissle 1917. <br />
</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/c/c6/VerifiGEM-Logo.jpg/800px-VerifiGEM-Logo.jpg" width="900" height="200" /></div><br />
<p style="color:black;text-indent:30px;"><br />
Furthermore, in an effort to speed progress and information sharing between iGEM teams, government research organizations, and private research groups, we have proposed a system known as VerifiGEM that would allow for the quality control of BioBricks to be distributed across the entire iGEM community. Should our system be implemented, the overall quality and reliability of BioBricks will be improved greatly, at very little cost to individual iGEM teams. <br />
</p><br />
</div><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Perception Barriers to Bacterial Therapeutics</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
However, the removal of biological barriers to bacterial therapeutics alone is not sufficient to enable bacterial therapeutics to move into the drug development pipeline. Through the course of recent history, many high profile technologies, such as gene therapy or nanotechnology have been met with public skepticism and even fear, as the technologies failed to deliver on earlier promises. This prompted a constriction of available funding and subsequently impeded progress in those fields, an outcome that nanotechnology in particular is only just beginning to recover from.<br />
</p><br />
<p style="color:black;text-indent:30px;"><br />
We propose a model, adapted from Gartner Inc, which proposes that the disconnect between the expectations of the public and the realities of scientific research produces an initial "peak of inflated expectations" (and funding), that rapidly disappears as promised advances are delayed or do not work as planned (Figure 1). We believe that this "trough of disillusionment" is the cause of restrictions in funding and a general stall of scientific progress in a given field. We propose that the peak can be smoothed out, reducing the size of peak, but more importantly, eliminating the trough of disillusionment (Figure 2).<br />
</p><br />
<br />
What then, must we as synthetic biologists do to prevent this fate from befalling our own field of study? Certainly, scientists must perform a balancing act between reporting the advances they have made and making realistic conclusions. iGEM teams in particular have a unique opportunity to <br />
</p><br />
</div><br />
<br />
<br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Human Practices</div></b><br />
<br><br />
<br />
<p style="color:black;text-indent:30px;">Upon conception of this project, we realized that although hundreds of academic research projects and iGEM projects have been conducted in the realm of Health and Medicine, almost no engineered bacterial therapeutics have been brought to the clinic. We analyzed the hurdles and road ahead for bacterial synthetic biology-enabled therapeutics, compiling a thorough report with specific actions which iGEM teams in Health/Medicine can take to make their therapies more clinically tractable. This project directly informed our wet lab work, causing us to port our therapeutic system into a non-pathogenic, probiotic bacterial strain which is already used in human therapies today.</p><br />
<br />
<br />
<p style="color:black;text-indent:30px;">We hope our targeted therapeutic platform will allow other scientists and iGEM teams to target any cells they choose. In the near term, we are planning to test our cancer cell targeting/killing bacterial system in a mouse model and make a real impact on cancer research and therapy.</p><br />
</div><br />
</body><br />
</html></div>Qiaophttp://2012.igem.org/File:VerifiGEM-Logo.jpgFile:VerifiGEM-Logo.jpg2012-10-27T01:53:31Z<p>Qiaop: </p>
<hr />
<div></div>Qiaophttp://2012.igem.org/Team:Penn/HumanPracticesOverviewTeam:Penn/HumanPracticesOverview2012-10-27T01:52:04Z<p>Qiaop: </p>
<hr />
<div>{{:Team:Penn/Template/Site}}<br />
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.all{ width:1000px; margin:0 auto;}<br />
.ImageBorder {border: 3px solid #ffffff;margin: 0 0 20px 0;}<br />
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.bigbox{width:936px; padding:30px;background:#d7dce1;border:2px solid #708090;border-radius:10px;-moz-border-radius:10px;-webkit-border-radius:10px;color:black; font-size:14px; text-align:justify; margin:0 0 20px 0;}<br />
.smallbox{ margin:20px auto; width:600px; padding:40px;background:#d7dce1;border:2px solid #708090;border-radius:10px;-moz-border-radius:10px;-webkit-border-radius:10px; overflow:hidden; text-align:justify;}<br />
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</style><br />
<br><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Human Practices: Transitioning From the Bench to the Bedside</div></b><br />
<br><br />
<p style="text-align:justify;">Many previous iGEM teams have tried to implement a bacterial therapeutic as part of their project. Outside of iGEM, there has been a steady interest in engineering bacteria to become therapeutic vectors as well. However, the question that guided our human practices project was essentially: <b><br><p style="text-align:center">Why aren't bacterial therapeutics transitioning into clinical practice or even clinical trials? </p></p></b><br />
<p style="text-align:justify;"><br />
While there are certainly many barriers to bacterial therapeutics such as time and money, we hypothesize that iGEM teams, as a result of their unique positions as research and educational institutions, are positioned to address two major barriers to the adoption of bacterial therapeutics: biological barriers and perception barriers.<br />
</p><br />
<br><br />
</div><br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Biological Barriers to Bacterial Therapeutics</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
From a technological standpoint, there is a great deal of work that remains to be done before a bacterial therapeutic can enter the drug development pipeline. While many iGEM teams, including us, have helped set the groundwork for bacterial therapeutics, there are still some biological barriers to a bacterial therapeutic. We identified the immunogenicity of laboratory strains of <i>E. coli.</i> as a major biological barrier. We then investigated methods to decrease the immunogenicity of <i>E. coli.</i>, eventually choosing to port modules of our target drug delivery system into a non-immunogenic strain of <i>E. coli.</i>, Nissle 1917. <br />
</p><br />
<p style="color:black;text-indent:30px;"><br />
Furthermore, in an effort to speed progress and information sharing between iGEM teams, government research organizations, and private research groups, we have proposed a system known as VerifiGEM that would allow for the quality control of BioBricks to be distributed across the entire iGEM community. Should our system be implemented, the overall quality and reliability of BioBricks will be improved greatly, at very little cost to individual iGEM teams. <br />
</p><br />
</div><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Perception Barriers to Bacterial Therapeutics</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
However, the removal of biological barriers to bacterial therapeutics alone is not sufficient to enable bacterial therapeutics to move into the drug development pipeline. Through the course of recent history, many high profile technologies, such as gene therapy or nanotechnology have been met with public skepticism and even fear, as the technologies failed to deliver on earlier promises. This prompted a constriction of available funding and subsequently impeded progress in those fields, an outcome that nanotechnology in particular is only just beginning to recover from.<br />
</p><br />
<p style="color:black;text-indent:30px;"><br />
We propose a model, adapted from Gartner Inc, which proposes that the disconnect between the expectations of the public and the realities of scientific research produces an initial "peak of inflated expectations" (and funding), that rapidly disappears as promised advances are delayed or do not work as planned (Figure 1). We believe that this "trough of disillusionment" is the cause of restrictions in funding and a general stall of scientific progress in a given field. We propose that the peak can be smoothed out, reducing the size of peak, but more importantly, eliminating the trough of disillusionment (Figure 2).<br />
</p><br />
<br />
What then, must we as synthetic biologists do to prevent this fate from befalling our own field of study? Certainly, scientists must perform a balancing act between reporting the advances they have made and making realistic conclusions. iGEM teams in particular have a unique opportunity to <br />
</p><br />
</div><br />
<br />
<br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Human Practices</div></b><br />
<br><br />
<br />
<p style="color:black;text-indent:30px;">Upon conception of this project, we realized that although hundreds of academic research projects and iGEM projects have been conducted in the realm of Health and Medicine, almost no engineered bacterial therapeutics have been brought to the clinic. We analyzed the hurdles and road ahead for bacterial synthetic biology-enabled therapeutics, compiling a thorough report with specific actions which iGEM teams in Health/Medicine can take to make their therapies more clinically tractable. This project directly informed our wet lab work, causing us to port our therapeutic system into a non-pathogenic, probiotic bacterial strain which is already used in human therapies today.</p><br />
<br />
<br />
<p style="color:black;text-indent:30px;">We hope our targeted therapeutic platform will allow other scientists and iGEM teams to target any cells they choose. In the near term, we are planning to test our cancer cell targeting/killing bacterial system in a mouse model and make a real impact on cancer research and therapy.</p><br />
</div><br />
</body><br />
</html></div>Qiaophttp://2012.igem.org/Team:Penn/HumanPracticesOverviewTeam:Penn/HumanPracticesOverview2012-10-27T01:35:57Z<p>Qiaop: </p>
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<b><div class="name" align="center">Human Practices: Transitioning From the Bench to the Bedside</div></b><br />
<br><br />
<p style="text-align:justify;">Many previous iGEM teams have tried to implement a bacterial therapeutic as part of their project. Outside of iGEM, there has been a steady interest in engineering bacteria to become therapeutic vectors as well. However, the question that guided our human practices project was essentially: <b><br><p style="text-align:center">Why aren't bacterial therapeutics transitioning into clinical practice or even clinical trials? </p></p></b><br />
<p style="text-align:justify;"><br />
While there are certainly many barriers to bacterial therapeutics such as time and money, we hypothesize that iGEM teams, as a result of their unique positions as research and educational institutions, are positioned to address two major barriers to the adoption of bacterial therapeutics: biological barriers and perception barriers.<br />
</p><br />
<br><br />
</div><br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Biological Barriers to Bacterial Therapeutics</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
From a technological standpoint, there is a great deal of work that remains to be done before a bacterial therapeutic can enter the drug development pipeline. While many iGEM teams, including us, have helped set the groundwork for bacterial therapeutics, there are still some biological barriers to a bacterial therapeutic. We identified the immunogenicity of laboratory strains of <i>E. coli.</i> as a major biological barrier. We then investigated methods to decrease the immunogenicity of <i>E. coli.</i>, eventually choosing to port modules of our target drug delivery system into a non-immunogenic strain of <i>E. coli.</i>, Nissle 1917. <br />
</p><br />
<p style="color:black;text-indent:30px;"><br />
Furthermore, in an effort to speed progress and information sharing between iGEM teams, government research organizations, and private research groups, we have proposed a system known as VerifiGEM that would allow for the quality control of BioBricks to be distributed across the entire iGEM community. Should our system be implemented, the overall quality and reliability of BioBricks will be improved greatly, at very little cost to individual iGEM teams. <br />
</p><br />
</div><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Perception Barriers to Bacterial Therapeutics</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
However, the removal of biological barriers to bacterial therapeutics alone is not sufficient to enable bacterial therapeutics to move into the drug development pipeline. Through the course of recent history, many high profile technologies, such as gene therapy or nanotechnology have been met with public skepticism and even fear, as the technologies failed to deliver on earlier promises.<br />
</p><br />
</div><br />
<br />
<br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Human Practices</div></b><br />
<br><br />
<br />
<p style="color:black;text-indent:30px;">Upon conception of this project, we realized that although hundreds of academic research projects and iGEM projects have been conducted in the realm of Health and Medicine, almost no engineered bacterial therapeutics have been brought to the clinic. We analyzed the hurdles and road ahead for bacterial synthetic biology-enabled therapeutics, compiling a thorough report with specific actions which iGEM teams in Health/Medicine can take to make their therapies more clinically tractable. This project directly informed our wet lab work, causing us to port our therapeutic system into a non-pathogenic, probiotic bacterial strain which is already used in human therapies today.</p><br />
<br />
<br />
<p style="color:black;text-indent:30px;">We hope our targeted therapeutic platform will allow other scientists and iGEM teams to target any cells they choose. In the near term, we are planning to test our cancer cell targeting/killing bacterial system in a mouse model and make a real impact on cancer research and therapy.</p><br />
</div><br />
</body><br />
</html></div>Qiaophttp://2012.igem.org/Team:Penn/HumanPracticesOverviewTeam:Penn/HumanPracticesOverview2012-10-27T01:34:46Z<p>Qiaop: </p>
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<b><div class="name" align="center">Human Practices: Transitioning From the Bench to the Bedside</div></b><br />
<br><br />
<p style="text-align:justify;">Many previous iGEM teams have tried to implement a bacterial therapeutic as part of their project. Outside of iGEM, there has been a steady interest in engineering bacteria to become therapeutic vectors as well. However, the question that guided our human practices project was essentially: <b><br><p style="text-align:center">Why aren't bacterial therapeutics transitioning into clinical practice or even clinical trials? </p></p></b><br />
<p style="text-align:justify;"><br />
While there are certainly many barriers to bacterial therapeutics such as time and money, we hypothesize that iGEM teams, as a result of their unique positions as research and educational institutions, are positioned to address two major barriers to the adoption of bacterial therapeutics: biological barriers and perception barriers.<br />
</p><br />
<br><br />
</div><br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Biological Barriers to Bacterial Therapeutics</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
From a technological standpoint, there is a great deal of work that remains to be done before a bacterial therapeutic can enter the drug development pipeline. While many iGEM teams, including us, have helped set the groundwork for bacterial therapeutics, there are still some biological barriers to a bacterial therapeutic. We identified the immunogenicity of laboratory strains of <i>E. coli.</i> as a major biological barrier. We then investigated methods to decrease the immunogenicity of <i>E. coli.</i>, eventually choosing to port modules of our target drug delivery system into a non-immunogenic strain of <i>E. coli.</i>, Nissle 1917. Furthermore, in an effort to speed progress and information sharing between iGEM teams, government research organizations, and private research groups, we have proposed a system that would allow for the quality control of BioBricks to be distributed across the entire iGEM community. Should our system be implemented, the overall quality and reliability of BioBricks will be improved greatly, at very little cost to individual iGEM teams. <br />
</p><br />
</div><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Perception Barriers to Bacterial Therapeutics</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
However, the removal of biological barriers to bacterial therapeutics alone is not sufficient to enable bacterial therapeutics to move into the drug development pipeline. Through the course of recent history, many high profile technologies, such as gene therapy or nanotechnology have been met with public skepticism and even fear, as the technologies failed to deliver on earlier promises.<br />
</p><br />
</div><br />
<br />
<br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Human Practices</div></b><br />
<br><br />
<br />
<p style="color:black;text-indent:30px;">Upon conception of this project, we realized that although hundreds of academic research projects and iGEM projects have been conducted in the realm of Health and Medicine, almost no engineered bacterial therapeutics have been brought to the clinic. We analyzed the hurdles and road ahead for bacterial synthetic biology-enabled therapeutics, compiling a thorough report with specific actions which iGEM teams in Health/Medicine can take to make their therapies more clinically tractable. This project directly informed our wet lab work, causing us to port our therapeutic system into a non-pathogenic, probiotic bacterial strain which is already used in human therapies today.</p><br />
<br />
<br />
<p style="color:black;text-indent:30px;">We hope our targeted therapeutic platform will allow other scientists and iGEM teams to target any cells they choose. In the near term, we are planning to test our cancer cell targeting/killing bacterial system in a mouse model and make a real impact on cancer research and therapy.</p><br />
</div><br />
</body><br />
</html></div>Qiaophttp://2012.igem.org/Team:Penn/NissleTeam:Penn/Nissle2012-10-27T01:34:20Z<p>Qiaop: </p>
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<b><div class="name" align="center">Use of the GRAS (Generally Regarded As Safe) Strain E. Coli Nissle 1917</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"> We have seen how the complex interplay between public opinion and science innovation can drastically affect the adoption and success of a new technology, such as our team's bacterial drug delivery system. As pointed out earlier, pervading public opinion towards a bacterial therapeutic system such as the one developed by our team this year would most likely be negative. In an effort to address some of these issues, we have set out to investigate ways our system could be made more palatable to the general public. </p><br />
<p style="color:black;text-indent:30px;"> One recent concept that we have identified as gaining general acceptance within the general public is the incorporation of "probiotic" organisms into a daily diet. Many foods, such as yogurts now advertise the presence of "probiotic" bacteria and there are "probiotic" supplements containing live bacterial cultures as well. One particular probiotic, E. Coli Nissle 1917 has attracted attention not only from the public, but also from the scientific community, where its potential beneficial properties have been investigated. The Nissle strain is notable for its lack of virulence factors and decreased immunogenecity [1]. These traits are what make Nissle a popular probiotic. Nissle has also been found to preferentially colonize tumors, proliferating wildly in the borders between live and necrotic tissue, a highly desirable trait for any potential cancer treatment [2]. Additional investigation has demonstrated that intravenously administered Nissle exhibits a similar behavior in breast cancer mouse models, and expression of recombinant azurin prevented cancer metastasis in mice [4]. However, the therapeutic potential of Nissle is not limited to cancer treatment. Nissle is also capable of enhancing wound healing through recombinant expression of human epidermal Growth Factor in the epithelial linings of the body, as well as reducing modulating responses to allergens [5,6].</p><br />
<p style="color:black;text-indent:30px;"> Based on these properties, we believe that demonstrating that our drug delivery system can be implemented in Nissle 1917 would be the first step to addressing the potential hesitance that the public may have to bacterial based therapies. Because the chassis for our system is a probiotic, we can avoid not only the technical difficulties of ensuring that the host for our system is inherently safe, but also proactively address (or at least minimize) the initial "knee-jerk" reactions that many members of the general public may have to the idea of a bacterial therapeutic. Furthermore, In order to fully realize the potential of Nissle, it is important to be able to easily and consistently manipulate and change its genetic information. Therefore, we have produced and characterized a process for generating chemically competent Nissle 1917 that can be produced in any standard microbiology lab, allowing future iGEM teams to unlock Nissle 1917's full potential.</p><div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/9/90/20121004033558!IMG_3309.JPG" width="600" height="400"></div><br />
</div><br />
<div class="bigbox"><br />
<b><div class="name" align="center">Production of Chemically Competent Nissle 1917 Cells</div></b><br />
<ol><br />
<li>Inoculate one colony from LB plate into 2 ml LB liquid medium. Shake at 37 °C<br />
overnight.</li><br />
<li>Inoculate 1-ml overnight cell culture into 100 ml LB medium (in a 500 ml flask).</li><br />
<li>Shake vigorously at 37 °C to OD600 ~0.25-0.3.</li><br />
<li>Chill the culture on ice for 15 min. Also make sure the 0.1M CaCl2<br />
solution and 0.1M CaCl2 plus 15% glycerol are on ice.</li><br />
<li>Centrifuge the cells for 10 min at 5000 g at 4°C.</li><br />
<li>Discard the medium and resuspend the cell pellet in 30-40 ml cold 0.1M CaCl2. Keep the cells on ice for 30 min.</li><br />
<li>Centrifuge the cells as above.</li><br />
<li>Remove the supernatant, and resuspend the cell pellet in 6 ml 0.1 M CaCl2<br />
solution plus 15% glycerol.</li><br />
<li>Pipet 0.4-0.5 ml of the cell suspension into sterile 1.5 ml micro-centrifuge tubes. Flash freeze these tubes in liquid nitrogen and then transfer them to the -80 C freezer.<br />
<ul><br />
<li><br />
Note: Successful transformations have occured with 100uL of cells + 1ug of DNA, however the efficency of cells made through this process is lower than that of Subcloning Efficency DH5a from Invitrogen. After flash freezing, competency of cells prepared through this protocol increases over time with additional storage time in -80°C for approximately 3 days.<br />
</li><br />
</ul><br />
</ol><br />
</div><br />
<div class="bigbox"><br />
<b><div class="name" align="center">References:</div></b><br><br />
<br />
[1] Grozdanov, L., U. Zahringer, G. Blum-Oehler, L. Brade, A. Henne, Y. A. Knirel, U.Schombel, J. Schulze, U. Sonnenborn, G. Gottschalk, J. Hacker, E. T. Rietschel, and U. Dobrindt. "A Single Nucleotide Exchange in the Wzy Gene Is Responsible for the Semirough O6 Lipopolysaccharide Phenotype and Serum Sensitivity of Escherichia Coli Strain Nissle 1917." Journal of Bacteriology 184.21 (2002): 5912-925. Print.<br><br />
<br><br />
[2] Stritzker, J., S. Weibel, P. Hill, T. Oelschlaeger, W. Goebel, and A. Szalay.<br />
<br />
"Tumor-specific Colonization, Tissue Distribution, and Gene Induction by Probiotic Escherichia Coli Nissle 1917 in Live Mice." International Journal of Medical Microbiology 297.3 (2007): 151-62. 19 Apr. 2007. Web. 29 Sept. 2012.<br><br />
<br><br />
[3] Weise, Christin, Yan Zhu, Dennis Ernst, Anja A. Kühl, and Margitta Worm. "Oral<br />
<br />
Administration of Escherichia Coli Nissle 1917 Prevents Allergen-induced Dermatitis in Mice." Experimental Dermatology 20.10 (2011): 805-09. 11 July 2011. Web. 29 Sept. 2012. <br><br />
<br><br />
[4] Zhang, Y., L. Xia, X. Zhang, X. Ding, F. Yan, and F. Wu. "Escherichia Coli Nissle 1917 Targets and Restrains Mouse B16 Melanoma and 4T1 Breast Tumor through the Expression of Azurin Protein." Applied Environmental Microbiology (n.d.): n. pag. 24 Aug. 2012. Web. 29 Sept. 2012.</div><br />
</html></div>Qiaophttp://2012.igem.org/Team:Penn/HumanPracticesOverviewTeam:Penn/HumanPracticesOverview2012-10-27T01:18:22Z<p>Qiaop: </p>
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<b><div class="name" align="center">Human Practices: Transitioning From the Bench to the Bedside</div></b><br />
<br><br />
<p style="text-align:justify;">Many previous iGEM teams have tried to implement a bacterial therapeutic as part of their project. Outside of iGEM, there has been a steady interest in engineering bacteria to become therapeutic vectors as well. However, the question that guided our human practices project was essentially: <b>Why aren't bacterial therapeutics transitioning into clinical practice or even clinical trials? </p></b><br />
<p style="text-align:justify;"><br />
While there are certainly many barriers to bacterial therapeutics such as time and money, we hypothesize that iGEM teams, as a result of their unique positions as research and educational institutions, are positioned to address two major barriers to the adoption of bacterial therapeutics: biological barriers and perception barriers.<br />
</p><br />
<br><br />
</div><br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Biological Barriers to Bacterial Therapeutics</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
From a technological standpoint, there is a great deal of work that remains to be done before a bacterial therapeutic can enter the drug development pipeline. While many iGEM teams, including us, have helped set the groundwork for bacterial therapeutics, there are still some biological barriers to a bacterial therapeutic. We identified the immunogenicity of laboratory strains of <i>E. coli.</i> as a major biological barrier. We then investigated methods to decrease the immunogenicity of <i>E. coli.</i>, eventually choosing to port modules of our target drug delivery system into a non-immunogenic strain of <i>E. coli.</i>, Nissle 1917. Furthermore, in an effort to speed progress and information sharing between iGEM teams, government research organizations, and private research groups, we have proposed a system that would allow for the quality control of BioBricks to be distributed across the entire iGEM community. Should our system be implemented, the overall quality and reliability of BioBricks will be improved greatly, at very little cost to individual iGEM teams. <br />
</p><br />
</div><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Perception Barriers to Bacterial Therapeutics</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
However, the removal of biological barriers to bacterial therapeutics alone is not sufficient to <br />
</p><br />
</div><br />
<br />
<br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Human Practices</div></b><br />
<br><br />
<br />
<p style="color:black;text-indent:30px;">Upon conception of this project, we realized that although hundreds of academic research projects and iGEM projects have been conducted in the realm of Health and Medicine, almost no engineered bacterial therapeutics have been brought to the clinic. We analyzed the hurdles and road ahead for bacterial synthetic biology-enabled therapeutics, compiling a thorough report with specific actions which iGEM teams in Health/Medicine can take to make their therapies more clinically tractable. This project directly informed our wet lab work, causing us to port our therapeutic system into a non-pathogenic, probiotic bacterial strain which is already used in human therapies today.</p><br />
<br />
<br />
<p style="color:black;text-indent:30px;">We hope our targeted therapeutic platform will allow other scientists and iGEM teams to target any cells they choose. In the near term, we are planning to test our cancer cell targeting/killing bacterial system in a mouse model and make a real impact on cancer research and therapy.</p><br />
</div><br />
</body><br />
</html></div>Qiaophttp://2012.igem.org/Team:Penn/HumanPracticesOverviewTeam:Penn/HumanPracticesOverview2012-10-27T01:18:07Z<p>Qiaop: </p>
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<div class="bigbox"><br />
<b><div class="name" align="center">Human Practices: Transitioning From the Bench to the Bedside</div></b><br />
<br><br />
<p style="text-align:justify;">Many previous iGEM teams have tried to implement a bacterial therapeutic as part of their project. Outside of iGEM, there has been a steady interest in engineering bacteria to become therapeutic vectors as well. However, the question that guided our human practices project was essentially: <b>Why aren't bacterial therapeutics transitioning into clinical practice or even clinical trials? </p></b><br />
<p style="text-align:justify;"><br />
While there are certainly many barriers to bacterial therapeutics such as time and money, we hypothesize that iGEM teams, as a result of their unique positions as research and educational institutions, are positioned to address two major barriers to the adoption of bacterial therapeutics: biological barriers and perception barriers.<br />
</p><br />
<br><br />
</div><br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Biological Barriers to Bacterial Therapeutics</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
From a technological standpoint, there is a great deal of work that remains to be done before a bacterial therapeutic can enter the drug development pipeline. While many iGEM teams, including us, have helped set the groundwork for bacterial therapeutics, there are still some biological barriers to a bacterial therapeutic. We identified the immunogenicity of laboratory strains of <i>E. coli.</i> as a major biological barrier. We then investigated methods to decrease the immunogenicity of <i>E. coli.</i>, eventually choosing to port modules of our target drug delivery system into a non-immunogenic strain of <i>E. coli.<i>, Nissle 1917. Furthermore, in an effort to speed progress and information sharing between iGEM teams, government research organizations, and private research groups, we have proposed a system that would allow for the quality control of BioBricks to be distributed across the entire iGEM community. Should our system be implemented, the overall quality and reliability of BioBricks will be improved greatly, at very little cost to individual iGEM teams. <br />
</p><br />
</div><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Perception Barriers to Bacterial Therapeutics</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
However, the removal of biological barriers to bacterial therapeutics alone is not sufficient to <br />
</p><br />
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<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Human Practices</div></b><br />
<br><br />
<br />
<p style="color:black;text-indent:30px;">Upon conception of this project, we realized that although hundreds of academic research projects and iGEM projects have been conducted in the realm of Health and Medicine, almost no engineered bacterial therapeutics have been brought to the clinic. We analyzed the hurdles and road ahead for bacterial synthetic biology-enabled therapeutics, compiling a thorough report with specific actions which iGEM teams in Health/Medicine can take to make their therapies more clinically tractable. This project directly informed our wet lab work, causing us to port our therapeutic system into a non-pathogenic, probiotic bacterial strain which is already used in human therapies today.</p><br />
<br />
<br />
<p style="color:black;text-indent:30px;">We hope our targeted therapeutic platform will allow other scientists and iGEM teams to target any cells they choose. In the near term, we are planning to test our cancer cell targeting/killing bacterial system in a mouse model and make a real impact on cancer research and therapy.</p><br />
</div><br />
</body><br />
</html></div>Qiaophttp://2012.igem.org/Team:Penn/HumanPracticesOverviewTeam:Penn/HumanPracticesOverview2012-10-27T01:12:17Z<p>Qiaop: </p>
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<b><div class="name" align="center">Human Practices: Transitioning From the Bench to the Bedside</div></b><br />
<br><br />
<p style="text-align:justify;">Many previous iGEM teams have tried to implement a bacterial therapeutic as part of their project. Outside of iGEM, there has been a steady interest in engineering bacteria to become therapeutic vectors as well. However, the question that guided our human practices project was essentially: <b>Why aren't bacterial therapeutics transitioning into clinical practice or even clinical trials? </p></b><br />
<p style="text-align:justify;"><br />
While there are certainly many barriers to bacterial therapeutics such as time and money, we hypothesize that iGEM teams, as a result of their unique positions as research and educational institutions, are positioned to address two major barriers to the adoption of bacterial therapeutics: biological barriers and perception barriers.<br />
</p><br />
<br><br />
</div><br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Biological Barriers to Bacterial Therapeutics</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
From a technological standpoint, there is a great deal of work that remains to be done before a bacterial therapeutic can enter the drug development pipeline. While many iGEM teams, including us, have helped set the groundwork for bacterial therapeutics, there are still some biological barriers to a bacterial therapeutic. We identified the immunogenicity of laboratory strains of <i>E. coli.</i> as a major biological barrier. We then investigated methods to decrease the immunogenicity of <i>E. coli.</i>, eventually choosing to port modules of our target drug delivery system into a non-immunogenic strain of <i>E. coli.<i>, Nissle 1917. <br />
</p><br />
</div><br />
<br />
<br />
<br />
<br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Human Practices</div></b><br />
<br><br />
<br />
<p style="color:black;text-indent:30px;">Upon conception of this project, we realized that although hundreds of academic research projects and iGEM projects have been conducted in the realm of Health and Medicine, almost no engineered bacterial therapeutics have been brought to the clinic. We analyzed the hurdles and road ahead for bacterial synthetic biology-enabled therapeutics, compiling a thorough report with specific actions which iGEM teams in Health/Medicine can take to make their therapies more clinically tractable. This project directly informed our wet lab work, causing us to port our therapeutic system into a non-pathogenic, probiotic bacterial strain which is already used in human therapies today.</p><br />
<br />
<br />
<p style="color:black;text-indent:30px;">We hope our targeted therapeutic platform will allow other scientists and iGEM teams to target any cells they choose. In the near term, we are planning to test our cancer cell targeting/killing bacterial system in a mouse model and make a real impact on cancer research and therapy.</p><br />
</div><br />
</body><br />
</html></div>Qiaophttp://2012.igem.org/Team:Penn/HumanPracticesOverviewTeam:Penn/HumanPracticesOverview2012-10-27T01:11:43Z<p>Qiaop: </p>
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<div class="bigbox"><br />
<b><div class="name" align="center">Human Practices: Transitioning From the Bench to the Bedside</div></b><br />
<br><br />
<p style="text-align:justify;">Many previous iGEM teams have tried to implement a bacterial therapeutic as part of their project. Outside of iGEM, there has been a steady interest in engineering bacteria to become therapeutic vectors as well. However, the question that guided our human practices project was essentially: <b>Why aren't bacterial therapeutics transitioning into clinical practice or even clinical trials? </p></b><br />
<p style="text-align:justify;"><br />
While there are certainly many barriers to bacterial therapeutics such as time and money, we hypothesize that iGEM teams, as a result of their unique positions as research and educational institutions, are positioned to address two major barriers to the adoption of bacterial therapeutics: biological barriers and perception barriers.<br />
</p><br />
<br><br />
</div><br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Biological Barriers to Bacterial Therapeutics</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
From a technological standpoint, there is a great deal of work that remains to be done before a bacterial therapeutic can enter the drug development pipeline. While many iGEM teams, including us, have helped set the groundwork for bacterial therapeutics, there are still some biological barriers to a bacterial therapeutic. We identified the immunogenicity of laboratory strains of <i>E. coli.</i> as a major biological barrier. We then investigated methods to decrease the immunogenicity of <i>E. coli.</i>, eventually choosing to port modules of our target drug delivery system into a non-immunogenic strain of <i>E. coli.<i>, Nissle 1917. <br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Human Practices</div></b><br />
<br><br />
<br />
<p style="color:black;text-indent:30px;">Upon conception of this project, we realized that although hundreds of academic research projects and iGEM projects have been conducted in the realm of Health and Medicine, almost no engineered bacterial therapeutics have been brought to the clinic. We analyzed the hurdles and road ahead for bacterial synthetic biology-enabled therapeutics, compiling a thorough report with specific actions which iGEM teams in Health/Medicine can take to make their therapies more clinically tractable. This project directly informed our wet lab work, causing us to port our therapeutic system into a non-pathogenic, probiotic bacterial strain which is already used in human therapies today.</p><br />
<br />
<br />
<p style="color:black;text-indent:30px;">We hope our targeted therapeutic platform will allow other scientists and iGEM teams to target any cells they choose. In the near term, we are planning to test our cancer cell targeting/killing bacterial system in a mouse model and make a real impact on cancer research and therapy.</p><br />
</div><br />
</body><br />
</html></div>Qiaophttp://2012.igem.org/Team:Penn/HumanPracticesOverviewTeam:Penn/HumanPracticesOverview2012-10-27T01:08:50Z<p>Qiaop: </p>
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<b><div class="name" align="center">Human Practices: Transitioning From the Bench to the Bedside</div></b><br />
<br><br />
<p style="text-align:justify;">Many previous iGEM teams have tried to implement a bacterial therapeutic as part of their project. Outside of iGEM, there has been a steady interest in engineering bacteria to become therapeutic vectors as well. However, the question that guided our human practices project was essentially: <b>Why aren't bacterial therapeutics transitioning into clinical practice or even clinical trials? </p></b><br />
<p style="text-align:justify;"><br />
While there are certainly many barriers to bacterial therapeutics such as time and money, we hypothesize that iGEM teams, as a result of their unique positions as research and educational institutions, are positioned to address two major barriers to the adoption of bacterial therapeutics: biological barriers and perception barriers.<br />
</p><br />
<br><br />
<br />
<div class="bigbox">Biological Barriers to Bacterial Therapeutics</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
From a technological standpoint, there is a great deal of work that remains to be done before a bacterial therapeutic can enter the drug development pipeline. While many iGEM teams, including us, have helped set the groundwork for bacterial therapeutics, there are still some biological barriers to a bacterial therapeutic. We identified the immunogenicity of laboratory strains of <i>E. coli.</i> as a major biological barrier. We then investigated methods to decrease the immunogenicity of <i>E. coli.</i>, eventually choosing to port modules of our target drug delivery system into a non-immunogenic strain of <i>E. coli.<i>, Nissle 1917. <br />
</p><br />
<br />
<p style="color:black;text-indent:30px;">The 2012 Penn iGEM team has engineered a novel platform for targeted therapeutics which employs simultaneous spatial and cellular targeting. We have achieved spatial (and temporal) targeting with a blue light-switchable transgene expression system, and cellular targeting through display of an antibody-mimetic protein on the surface of E. coli for the first time.</p><br />
<br />
<p style="color:black;text-indent:30px;">As a proof of concept, we applied our system to the treatment of cancer, a disease in which spatial and cellular targeting are of utmost importance. We displayed a high-affinity antibody-mimetic protein which targets Human Epidermal Growth Factor Receptor 2 (HER2), a protein commonly overexpressed in cancer cells. We combined this cellular targeting with a light-activated cytotoxic protein delivery system to successfully target and kill breast cancer cells.</p><br />
</div><br />
<br />
<br />
<br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Human Practices</div></b><br />
<br><br />
<br />
<p style="color:black;text-indent:30px;">Upon conception of this project, we realized that although hundreds of academic research projects and iGEM projects have been conducted in the realm of Health and Medicine, almost no engineered bacterial therapeutics have been brought to the clinic. We analyzed the hurdles and road ahead for bacterial synthetic biology-enabled therapeutics, compiling a thorough report with specific actions which iGEM teams in Health/Medicine can take to make their therapies more clinically tractable. This project directly informed our wet lab work, causing us to port our therapeutic system into a non-pathogenic, probiotic bacterial strain which is already used in human therapies today.</p><br />
<br />
<br />
<p style="color:black;text-indent:30px;">We hope our targeted therapeutic platform will allow other scientists and iGEM teams to target any cells they choose. In the near term, we are planning to test our cancer cell targeting/killing bacterial system in a mouse model and make a real impact on cancer research and therapy.</p><br />
</div><br />
</body><br />
</html></div>Qiaophttp://2012.igem.org/Team:Penn/HumanPracticesOverviewTeam:Penn/HumanPracticesOverview2012-10-27T00:50:09Z<p>Qiaop: </p>
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<b><div class="name" align="center">Human Practices: Transitioning From the Bench to the Bedside</div></b><br />
<br><br />
<p style="text-align:justify;">Many previous iGEM teams have tried to implement a bacterial therapeutic as part of their project. Outside of iGEM, there has been a steady interest in engineering bacteria to become therapeutic vectors as well. However, the question that guided our human practices project was essentially: <b>Why aren't bacterial therapeutics transitioning into clinical practice or even clinical trials? </p></b><br />
<p style="text-align:justify;"><br />
While there are certainly many barriers to bacterial therapeutics such as time and money, we hypothesize that iGEM teams, as a result of their unique positions as research and educational institutions, are positioned to address two major barriers to the adoption of bacterial therapeutics: biological barriers and perception barriers.<br />
</p><br />
<br><br />
<div align="center"><br />
<table width="860" cellspacing="20" style="background-color:#d7dce1;"><br />
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<td width="410"><img src="https://static.igem.org/mediawiki/2012/f/fa/Spatial_Targeting.jpg" width="400" height="300" /><br />
</td><br />
<td width="410"><img src="https://static.igem.org/mediawiki/2012/6/6b/Cellular_Targeting.jpg" width = "400" height = "300" /><br />
</td><br />
</tr><br />
<tr valign="top"><br />
<td ><br />
<p style="text-align:justify;"><b>Spatial Targeting:</b> Surgeons excise a tumor manually, without regard for cellular heterogeneity within and around the tumor area.</p><br />
</td><br />
<td><br />
<p style="text-align:justify;"><b>Cellular Targeting:</b> Monoclonal antibodies identify antigens on certain cells or viruses. Monoclonal antibodies are often coupled with therapeutic agents. However, if the antigen is present in healthy tissue outside the diseased area, it will be targeted as well.</p><br />
</td><br />
</tr><br />
<br />
</table></div><br />
<br />
These targeting mechanisms are imperfect on their own because they also target healthy tissue. Furthermore, the majority of therapies employ no targeting mechanisms at all (e.g. pharmacologic therapies). <br />
Even when diseases are clearly localized in specific areas and specific cells (such as cancer), current therapies such as chemotherapy attack the entire body and result in significant adverse effects. Patients who undergo chemotherapy suffer significant damage to fast-dividing cells throughout the entire body, which can result in immune system depression, hair loss, pain, and organ damage.<br />
</div><br />
<div class="bigbox"><br />
<b><div class="name" align="center">A Novel Therapeutic Platform</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;">What if you could combine spatial targeting and cellular targeting into the same therapeutic? This idea is unprecedented but would allow for precise targeting of specific cells within a specific area, leaving healthy tissue intact and keeping side effects to a minimum.</p><br />
<br />
<p style="color:black;text-indent:30px;">The 2012 Penn iGEM team has engineered a novel platform for targeted therapeutics which employs simultaneous spatial and cellular targeting. We have achieved spatial (and temporal) targeting with a blue light-switchable transgene expression system, and cellular targeting through display of an antibody-mimetic protein on the surface of E. coli for the first time.</p><br />
<br />
<p style="color:black;text-indent:30px;">As a proof of concept, we applied our system to the treatment of cancer, a disease in which spatial and cellular targeting are of utmost importance. We displayed a high-affinity antibody-mimetic protein which targets Human Epidermal Growth Factor Receptor 2 (HER2), a protein commonly overexpressed in cancer cells. We combined this cellular targeting with a light-activated cytotoxic protein delivery system to successfully target and kill breast cancer cells.</p><br />
</div><br />
<br />
<br />
<br />
<div class="bigbox"><br />
<br />
<b><div class="name" align="center">Human Practices</div></b><br />
<br><br />
<br />
<p style="color:black;text-indent:30px;">Upon conception of this project, we realized that although hundreds of academic research projects and iGEM projects have been conducted in the realm of Health and Medicine, almost no engineered bacterial therapeutics have been brought to the clinic. We analyzed the hurdles and road ahead for bacterial synthetic biology-enabled therapeutics, compiling a thorough report with specific actions which iGEM teams in Health/Medicine can take to make their therapies more clinically tractable. This project directly informed our wet lab work, causing us to port our therapeutic system into a non-pathogenic, probiotic bacterial strain which is already used in human therapies today.</p><br />
<br />
<br />
<p style="color:black;text-indent:30px;">We hope our targeted therapeutic platform will allow other scientists and iGEM teams to target any cells they choose. In the near term, we are planning to test our cancer cell targeting/killing bacterial system in a mouse model and make a real impact on cancer research and therapy.</p><br />
</div><br />
</body><br />
</html></div>Qiaophttp://2012.igem.org/Team:Penn/OutreachTeam:Penn/Outreach2012-10-27T00:40:54Z<p>Qiaop: </p>
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<b><div class="name" align="center">Clark Park Science Discovery Day</div></b><br><br />
As part of Science Discovery Day, the 2012 Penn iGEM team engaged with coordinators of the 2012 Philadelphia Science Festival, a week-long scientific expo devoted to promoting scientific education and literacy. Penn iGEM 2012 prepared a rudimentary alcohol DNA extraction from human cheek epithelial cells for visitors to try. After a successful extraction, team member would explain our project, as well as answer any questions about the current state-of-the-art of synthetic biology, after which experimenters could leave with a sample of their DNA. On April 29th, the team traveled to Clark Park in West Philadelphia to spend an afternoon demonstrating basic synthetic biology techniques, educating adults and children about synthetic biology, and leading DNA extraction experiments for groups of future synthetic biologists. Over 100 participants visited the Penn iGEM table in just over five hours!<br />
</div><br />
<div class="bigbox"><br />
<b><div class="name" align="center">SEAS SAAST 2012</div></b><br><br />
Penn iGEM 2012 has a continued dedication to scientific outreach and synthetic biology education. To further promote understanding of synthetic biology, the 2012 team presented to high school students participating in the University Of Pennsylvania School Of Engineering and Applied Science's SAAST (Summer Academy in Applied Science &amp; Technology) program. The SAAST program brings together over 170 high school kids for a two week long overview of engineering disciplines, where they attend compressed versions of engineering courses offered to undergraduates in the engineering school, as well as participate in practical laboratory investigations. As part of the biotechnology rotation, Penn iGEM 2012 was invited to present our research to the students and answer any questions they would have in the iconic Wu &amp; Chen auditorium on Penn's campus.<br />
</div><br><br />
<h1 align="center" style="color:white;"> Photos From The Events! </h1><br />
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<div style="background-color:white;padding-left:20px; padding-right:20px;"><br />
</p></div>Qiaophttp://2012.igem.org/Team:Penn/BarriersTeam:Penn/Barriers2012-10-27T00:40:09Z<p>Qiaop: </p>
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<div style="text-align:center;font-size:34px;color:white;"><b>Overview of Bacterial Therapeutics</b></div><br><br />
<br />
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<p style="color:black;text-indent:30px;">Currently in the field of medicine, groundbreaking approaches to therapeutics are continually being developed. With the advent of synthetic biology, bacterial therapy is rapidly emerging as a promising source of therapeutic potential for many diseases, especially cancer. Consequently, bacteria can actually be seen as the ideal vector for the production and delivery of drugs for many diseases.</p><br />
<br />
<b><div class="name" align="center">Why Bacteria are Ideal</div></b><br><br />
<p style="color:black;text-indent:30px;"><br />
One reason bacterial therapies have been proven to be better than many existing therapies is due to the fact that many bacteria already exist in a commensal or even mutualistic relationship with the human body. The reason this serves as an important advantage over other options is that it lessens the chance of a harmful response from the immune system. Introducing foreign entities into the body reduces the chance that the therapy would continue to work after a long period of time, since the body's defense mechanisms would kick in. This is exactly why working with or engineering strains that are part of the various microbiomes of the human body would be ideal. Escherichia coli is an example of a commensal bacteria in the gut that has been engineered to treat cholera. Scientists have managed to program E. coli to express cholera autoinducer 2 (CAI-2), inhabit the gut, and effectively block the quorum sensing system that cholera bacteria use to propagate disease in the gut [1]. Another example of this has been through engineering Lactobacillus L. jensenii, a vaginal strain to produce anti-HIV proteins to serve as a preventive measure again HIV [2].</p><br />
<p style="color:black;text-indent:30px;"><br />
Another feature that makes bacteria optimal is that they already contain compounds and metabolic pathways that can release or produce drugs. As a result, this helps reduce costs for extra materials by utilizing natural processes. Complete chemical synthesis is very costly, so any efforts that can harness the biosynthetic processes are greatly preferred. However, using a purely biosynthetic pathway leads to low yields. This is where synthetic biology techniques come into play where low prices and high yields can become a reality. A case where synthetic processes have been utilized effectively is in the case of the anti-malarial drug Artemisinin. Scientists have successfully managed to engineer E. coli to produce a precursor of Artemisinin by altering some pathways that can purified in a cost-effective manner [3]. Another great bacterium to engineer is Lactobacillus, due to the fact that it is part of pathway of fermenting milk into yogurt. Using this strategy, in 2008, MIT's iGEM team attempted to manipulate Lactobacillus bulgaricus to produce an antimicrobial peptide that would inhibit the binding of Streptococcus mutans. This would eventually prevent the formation of cavities. Moreover, this method effectively creates a cheaper, sustainable way to keep the therapy functional since small amounts of the bacteria are required to re-ferment milk and keep producing this antimicrobial yogurt[4].</p><p style="color:black;text-indent:30px;"><br />
In addition, a large proportion of diseases are bacterial. This allows one to consider the possible interplay between different bacteria. One way in which this interaction has been embraced is in the earlier cholera therapeutic with E. coli. Here we have two bacteria, engineered E. coli Nissle 1917 and Vibrio cholera, of which both have the ability to quorum sense or communicate with other cells. However, the latter is pathogenic and the former is not. As a result, since bacteria have the ability to communicate only with other bacteria that produce the specific CAI-2 it allows the E. coli to trick the Vibrio cholera to believe that the they have already inhabited that part of the gut and prevent the formation of the cholera biofilm [1]. These types of bacterial interactions are an interesting consideration to make when creating therapeutics where other non-pathogenic bacteria can beat pathogenic bacteria at their own game by emulating their behaviour.</p><p style="color:black;text-indent:30px;"><br />
Many bacteria are also facultative or obligate anaerobes. This is incredibly relevant for many diseases where hypoxic areas need to be targeted, the most obvious one being cancer. Initial attempts were done with facultative anaerobes (although considered to be an aerobe at the time) such as Listeria monocytogenes because they are noninvasive and are easy to control through antibiotics. However although they did cause some tumors to regress, ultimately they preferred aerobic environments [5]. Then they tried Salmonella, which was another facultative anaerobe, but thought to favor anaerobic environments more. Due to this preference they were predicted to accumulate around the necrotic centers of cancerous tumors, and especially after modifications made to amino acid machinery [6]. Nevertheless, it remained flawed due to the fact that it was invasive and the safety, and viability of the option in the long-term remained unpredictable. Finally, more recently, an attempt was made with Clostridium, an obligate anaerobe. This was a great candidate since it would target only oxygen-depleted areas of tumors, and thus not be able to cause any systemic disease. In addition, Clostridium are sporulating bacteria which enable them to be metabolically active only in areas of necrotic tissue. As a result, the Clostridium would be inert everywhere else and thus unlikely to cause an immune response [7]. When this was tried in practice, the localization of Clostridium tetani turned out to be highly concentrated in hypoxic areas and therefore made it a suitable target for cancer therapies [8]. Clostridium was eventually tried out years later as a vector and proved to have sustained anti-tumor effects [9].</p><p style="color:black;text-indent:30px;"><br />
Bacteria are also advantageous by the fact that they motile due to their flagella. This is especially useful in diseases where the area of infection is large and deep. The bacterial flagellum is useful in these situations because they have the ability to penetrate tissues [10]. Moreover, this also illustrates the point that bacteria will not be entropically limited, unlike passive molecules, and can thus acquire energy to reach areas that are not as easily accessible. This results in allowing the bacterial density to be higher in areas away from the vascular source from which they arrived. Once again this sort of advantage primes bacterial therapies well for areas of tumors that are quite large and deep. Furthermore, the flagella allow bacteria to exhibit chemotaxis which is once again beneficial for cancer therapies. Several bacteria have shown to accumulate preferentially in tumorous regions due to the nutrients provided from dying cells [11].</p><p style="color:black;text-indent:30px;"><br />
Additional points can be made to show why bacterial therapeutics are just plain convenient. First of all, bacterial genetics are very well-understood. This makes them easy to manipulate and build circuits in, similar to electrical engineering. In addition, bacteria are responsive to their environment which enables the potential to create "smarter" therapeutics that would enable precise controls even after delivery, and eventually ensure long-term success of the treatment. These controls can be achieved through creating circuits that are responsive to their environment using various promoter strategies. Finally, bacteria are very easily externally detectable through various techniques that include the use of light, magnetic resonance imaging, and positron emission tomography. This will enable scientists to measure the state of the tumor and therefore the success and efficacy of treatment.</p><br />
</div><br />
<div class="bigbox"><br />
<b><div class="name" align="center">Current State of Bacterial Therapy</div></b><br><br />
<p style="color:black;text-indent:30px;"><br />
However despite all these advantages, bacterial therapies have a low presence in the market, and engineered bacterial therapies are practically non-existent. One reason is that bacterial therapies often have trouble obtaining venture capital since many people still remain uncomfortable with putting genetically-modified organisms in their bodies. As a result, this essentially inhibits the commercialization of these products. But even before that, these therapies encounter additional resistance from the governmental agencies like the FDA and they can't even enter the market to begin with. Just this year, 2012, has Artemisinin, which comes from both genetically engineered bacteria and yeast become FDA-approved and ready to come in to the market soon. What's interesting is that these types of bacterial therapeutics, or at least probiotics widely used in Europe and Asia, but only recently has the market been rising in the United States [12]. Several companies such as Osel and Oragenics are companies that exclusively focus on developing bacterial therapeutics. Osel focuses primarily on harnessing the microbiomes already present in the human body. Although they have several probiotics in clinical trials none of their actual engineered probiotics have been able to go past the pre-clinical stage [13]. Oragenics focuses more on oral delivery of probiotics and has managed to bring a few products into the market.</p><p style="color:black;text-indent:30px;"><br />
Thus, by understanding these the various advantages bacterial therapies have, we hope to create two systems: a light-activated bacterial therapeutic and antimicrobial biofilm. However, also being aware that many of these bacterial therapeutic never make it into the clinic we hope to analyze why from a scientific, governmental, and social perspective to better understand the issue. Then based on this analysis offer some solutions that can streamline the process of eventually bringing these engineered bacterial systems into the clinic.</p><br />
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<div class="bigbox"><br />
<b><div class="name" align="center">Challenges Bacterial Therapy Faces</div></b><br><br />
<p style="color:black;text-indent:30px;"><br />
Although bacterial present themselves as ideal vehicles for drug delivery for various types of diseases there are several hurdles that need to be overcome before they become clinically viable. One major issue is the control of drug delivery. It is essential that the drug be delivered at high enough concentrations to sufficiently induce the appropriate therapeutic effects, yet low enough as to not be toxic to the body. [need to find existing solutions]<br />
The inherent toxicity of bacteria also presents an additional challenge. Although these engineered bacteria may appear to non-pathogenic in various healthy animal and human trials, their remaining virulence may still be able to affect patients who are immunocompromised, such as those with HIV and cancer. Scientists have attempted to engineer pathogenic strains bacteria such as Salmonella so that they may have decreased virulence. However, these efforts have not been successful in clinical trials [1].The bacteria remained too virulent to be used inside the human body due to their immunogenicity and pathogenicity outweighing any therapeutic effects[1]. As a result, scientists have been experimenting with nonpathogenic bacteria like Escherichia coli Nissle 1917. This strain that has been shown to be completely non-pathogenic not exporting any toxins. Scientists have been trying to engineer their various systems into this strain, however it has only been shown to be successful in situations where they behave commensally in the gut flora [2].</p><p style="color:black;text-indent:30px;"><br />
In addition, since bacterial therapeutics can be variable in their targeting efficiency they pose additional challenges. The problem is that cancer is unique in two ways. First, there are many types of cancer, each of which may have several subtypes. Each cancer subtype has a unique profile, meaning that a single bacterial therapeutic will interact differently with what may at first glance seem like the same type of disease. Secondly, each person's body will interact with the drug differently. Due to this uncertainty, the ideal dosage of the bacterial therapeutic varies between patients, making dosage decisions more difficult and less accurate. These ideas are what<br />
Moreover, this lack of consistent targeting efficiency will pose a larger problem for diseases that have metastatic potential. Not only won't the drug be able to spread uniformly, in the case of cancer, throughout the tumor, but also it won't be able to target more distal areas easily.</p><p style="color:black;text-indent:30px;"><br />
An additional issue is that the engineered bacteria may be genetically unstable, which may create mutations that alter the pathogenicity or growth potential of the cell itself.</p><p style="color:black;text-indent:30px;"><br />
Another problem is the method of delivery itself. Oral delivery of bacterial therapeutics forces bacteria to experience the harsh environment of the GI tract which causes low survival rates. This can be alleviated through higher dosages, but that can then cause an immunogenic response.</p><br />
</div><br />
<div class="bigbox"><br />
<b><div class="name" align="center">References</div></b><br><br />
<p style="color:black;text-indent:30px;"><br />
[1] Duan, F., & March, J. C. (2010). Engineered bacterial communication prevents Vibrio cholerae virulence in an infant mouse model. Proceedings of the National Academy of Sciences of the United States of America, 107(25), 11260–11264. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/20534565<br />
<p style="color:black;text-indent:30px;"><br />
[2] Liu, X., Lagenaur, L. A., Lee, P. P., & Xu, Q. (2008). Engineering of a human vaginal Lactobacillus strain for surface expression of two-domain CD4 molecules. Applied and Environmental Microbiology, 74(15), 4626–4635. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2504410&tool=pmcentrez&rendertype=abstract<br />
<p style="color:black;text-indent:30px;"><br />
[3] Tsuruta, H., Paddon, C. J., Eng, D., Lenihan, J. R., Horning, T., Anthony, L. C., Regentin, R., et al. (2009). High-Level Production of Amorpha-4,11-Diene, a Precursor of the Antimalarial Agent Artemisinin, in Escherichia coli. (A. Gregson, Ed.)PLoS ONE, 4(2), 12. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2637983&tool=pmcentrez&rendertype=abstract<br />
<p style="color:black;text-indent:30px;"><br />
[4] "Biogurt: A Sustainable and Savory Drug Delivery System." Main Page. MIT, n.d. Web. 02 Aug. 2012. <https://2008.igem.org/Team:MIT>.<br />
<p style="color:black;text-indent:30px;"><br />
[5] Pan, Z. K., Ikonomidis, G., Pardoll, D. & Paterson, Y. (1995). Regression of established tumors in mice mediated by the oral administration of a recombinant Listeria monocytogenes vaccine. Cancer Res 55, 4776-4779.<br />
<p style="color:black;text-indent:30px;"><br />
[6] Pawelek, J. M., Low, K. B. & Bermudes, D. (1997). Tumor-targeted Salmonella as a novel anticancer vector. Cancer Res 57, 4537-4544.<br />
<p style="color:black;text-indent:30px;"><br />
[7] Van Mellaert, L., Barbe, S. & Anne, J. (2006). Clostridium spores as anti-tumour agents. Trends Microbiol 14, 190-196.<br />
<p style="color:black;text-indent:30px;"><br />
[8] Lambin, P., Theys, J., Landuyt, W., Rijken, P., Van Der Kogel, A., Van Der Schueren, E., Hodgkiss, R., et al. (1998). Colonisation of Clostridium in the body is restricted to hypoxic and necrotic areas of tumours. Anaerobe, 4(4), 183–188. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/16887640<br />
<p style="color:black;text-indent:30px;"><br />
[9] Theys, J., Pennington, O., Dubois, L., Anlezark, G., Vaughan, T., Mengesha, A., Landuyt, W., et al. (2006). Repeated cycles of Clostridium-directed enzyme prodrug therapy result in sustained antitumour effects in vivo. British Journal of Cancer, 95(9), 1212–1219. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2360559&tool=pmcentrez&rendertype=abstract<br />
<p style="color:black;text-indent:30px;"><br />
[10] Toley, B. J., & Forbes, N. S. (2012). Motility is critical for effective distribution and accumulation of bacteria in tumor tissue. Integrative biology : quantitative biosciences from nano to macro, 4(2), 165–76. Retrieved from http://pubs.rsc.org/en/content/articlehtml/2012/ib/c2ib00091a<br />
<p style="color:black;text-indent:30px;"><br />
[11] . Kasinskas, R. W. & Forbes, N. S. Salmonella typhimurium specifically chemotax and proliferate in heterogeneous tumor tissue in vitro. Biotechnol. Bioeng. 94, 710–721 (2006).<br />
<p style="color:black;text-indent:30px;"><br />
[12] Starling, Shane. "Global Probiotics Market Approaching $30bn by 2015: Report."NutraIngredients.com. N.p., 15 Sept. 2010. Web. 03 Aug. 2012. <http://www.nutraingredients.com/Consumer-Trends/Global-probiotics-market-approaching-30bn-by-2015-Report>.<br />
<p style="color:black;text-indent:30px;"><br />
[13] "Clinical Development." Osel: Harnessing the Microbiome. N.p., n.d. Web. 03 Aug. 2012. <http://www.oselinc.com/clinical.html>.<br />
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<b><div class="name" align="center">Safety</div></b><br /><br />
<b class="blue"><br />
1. Would any of your project ideas raise safety issues in terms of:<br />
<ul class="grey"><br />
<li>researcher safety, </li><br />
<li>public safety, or </li><br />
<li>environmental safety? </li><br />
</ul><br />
</b><br />
<br />
<p><br />
<u><br />
Researcher safety<br />
</u><br />
</p><br />
<p style="color:black"><br />
&nbsp; &nbsp; The majority of our research is conducted with E. coli bacteria. E. coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of mammals. It is one of the most widely studied prokaryotes, and is used by scientists all over the world as a host organism when working with recombinant DNA, as well as for protein expression.<br />
</p><br />
<p style="color:black"><br />
&nbsp; &nbsp; The E. coli strains commonly used in the lab, such as DH5α and BL-21 bacteria, have been specifically engineered to be grown in a carefully regulated environment that only a laboratory can provide. Due to their sensitivity, they are unable to survive in the human body for an extended period of time. Consequently, the major risk of working with E. coli in the lab is not illness or infection, but rather contamination of other experiments such as eukaryotic cell culture. However, although the risk is deemed acceptable in labs, there is a strict autoclaving policy for all waste fluids, plates, and other waste produced when working with bacteria to mitigate risk to the general public if released by accident. Disposable items, such as tips, agar plates, and spreaders are incinerated, while glassware and other non-disposable equipment is autoclaved or cleaned with 70% isopropanol. Live cultures are treated with bleach using guidelines put forth by the Office of Environmental Health & Radiation Safety (EHRS). Furthermore, all sinks that are used to dispose of liquid waste are treated as potentially hazardous biological waste and are sterilized accordingly by the university.<br />
Additionally, these bacteria pose little risk to the environment. These strains of bacteria require conditions that would be unlikely to be found outside of the laboratory.<br />
</p><br />
<br />
<p style="color:black"><br />
<u><br />
Public Safety<br />
</u><br />
</p><br />
<p style="color:black"><br />
&nbsp; &nbsp; The greatest risk E. coli poses to the health and safety of the general public is through intentional, malicious use of the bacteria. While this is always a possibility with any project, our project does present a slightly increased risk due to our use of genes that confer antibiotic resistance as selection factors. This may increase the pathogenicity of the bacteria, but through our strict regulation of the use of these bacteria and stringent disposal guidelines, we believe we have adequately addressed this issue. Furthermore, access to our lab is strictly regulated, and it is extremely difficult for unauthorized individuals to access areas where E. coli is being stored or grown.<br />
</p><br />
<br />
<p style="color:black"><br />
<u><br />
Environmental safety<br />
</u><br />
</p><br />
<p style="color:black"><br />
&nbsp; &nbsp; The strains of bacteria that we use in our experiments pose little risk to the environment. This is mainly in part because these strains of bacteria require conditions that would be unlikely to be found outside of the laboratory.<br />
</p></div><br />
<div class="bigbox"><br />
<b class="blue"><br />
2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes,<br />
<ul class="grey"><br />
<li>Did you document these issues in the Registry?</li><br />
<li>How did you manage to handle the safety issue?</li><br />
<li>How could other teams learn from your experience?</li><br />
</ul><br />
</b><br />
<p style="color:black"><br />
&nbsp; &nbsp; While most of our devices and BioBrick parts do not pose any significant safety issues that have not already been addressed, we are planning to biobrick a cytotoxic protein known as clyA. ClyA variants have been used by iGEM teams in the past, however we were unable to determine what precautions they took when using the protein. We also evaluated literature where researchers used clyA, and again, could not find specific safety recommendations. Due to the lack of specific information, we performed all experiments involving clyA under BSL-2 precautions, which was the highest rating available to us. From our experience, we have found that the best resource when dealing with agents of unknown or unclear toxicity, such as clyA, is through the department responsible for laboratory safety at whichever institution the research is being performed. We plan to include the recommendations of our Biosafety Committee and our experiences with clyA in the Registry once our biobrick is submitted.<br />
<br />
</p></div><br />
<div class="bigbox"><br />
<b class="blue"><br />
3. Is there a local biosafety group, committee, or review board at your institution?<br />
<ul class="grey"><br />
<li>If yes, what does your local biosafety group think about your project?</li><br />
<li>If no, which specific biosafety rules or guidelines do you have to consider in your country?</li><br />
</ul><br />
</b><br />
<p style="color:black"><br />
&nbsp; &nbsp; As one of the world's foremost research institutions, the University of Pennsylvania has an extensive biological <a style="color:blue" href="http://www.ehrs.upenn.edu/programs/bio/bsm/">safety manual</a> that we abide by. Through these rules we have been approved for recombinant DNA lab work under a BSL-2 degsignation from the University EHRS after review. All members of our lab have completed a basic laboratory safety and biosafety program, as well as further training from postdocs and more experienced lab members. Currently our lab falls under the full BSL-2 designation for laboratories, which allows us to perform most of our work. Our mentor, Dr. Sarkar, has generously provided us with additional BSL-2 lab space that we can use to perform some of our other experiments. The BSL ratings and other biosafety regulations can be found <a href="http://www.ehrs.upenn.edu/programs/bio/bsm/principles.html" style="color:blue">here</a><br />
</p></div><br />
<div class="bigbox"><br />
<b class="blue"><br />
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?<br />
</b><br />
<br />
<p style="color:black"><br />
&nbsp; &nbsp; The current safeguards that most research institutions use are designed primarily to protect the researcher from exposure to organisms, DNA, and chemicals that may cause harm. However, there is still room for improvement when considering the control of recombinant DNA (rDNA). For example, while unlikely, there is a possibility that a plasmid, perhaps containing a virulence factor, toxin, or antibiotic resistance gene, could be transferred from a laboratory strain of bacteria to a more hardy strain without the knowledge of the researcher (horizontal gene transfer). Therefore, it may be useful for future iGEM teams to investigate ways that plasmids can be designed to minimize this risk. <br />
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<b><div class="name" align="center">Safety</div></b><br /><br />
<b class="blue"><br />
1. Would any of your project ideas raise safety issues in terms of:<br />
<ul class="grey"><br />
<li>researcher safety, </li><br />
<li>public safety, or </li><br />
<li>environmental safety? </li><br />
</ul><br />
</b><br />
<br />
<p><br />
<u><br />
Researcher safety<br />
</u><br />
</p><br />
<p style="color:black"><br />
&nbsp; &nbsp; The majority of our research is conducted with E. coli bacteria. E. coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of mammals. It is one of the most widely studied prokaryotes, and is used by scientists all over the world as a host organism when working with recombinant DNA, as well as for protein expression.<br />
</p><br />
<p style="color:black"><br />
&nbsp; &nbsp; The E. coli strains commonly used in the lab, such as DH5α and BL-21 bacteria, have been specifically engineered to be grown in a carefully regulated environment that only a laboratory can provide. Due to their sensitivity, they are unable to survive in the human body for an extended period of time. Consequently, the major risk of working with E. coli in the lab is not illness or infection, but rather contamination of other experiments such as eukaryotic cell culture. However, although the risk is deemed acceptable in labs, there is a strict autoclaving policy for all waste fluids, plates, and other waste produced when working with bacteria to mitigate risk to the general public if released by accident. Disposable items, such as tips, agar plates, and spreaders are incinerated, while glassware and other non-disposable equipment is autoclaved or cleaned with 70% isopropanol. Live cultures are treated with bleach using guidelines put forth by the Office of Environmental Health & Radiation Safety (EHRS). Furthermore, all sinks that are used to dispose of liquid waste are treated as potentially hazardous biological waste and are sterilized accordingly by the university.<br />
Additionally, these bacteria pose little risk to the environment. These strains of bacteria require conditions that would be unlikely to be found outside of the laboratory.<br />
</p><br />
<br />
<p style="color:black"><br />
<u><br />
Public Safety<br />
</u><br />
</p><br />
<p style="color:black"><br />
&nbsp; &nbsp; The greatest risk E. coli poses to the health and safety of the general public is through intentional, malicious use of the bacteria. While this is always a possibility with any project, our project does present a slightly increased risk due to our use of genes that confer antibiotic resistance as selection factors. This may increase the pathogenicity of the bacteria, but through our strict regulation of the use of these bacteria and stringent disposal guidelines, we believe we have adequately addressed this issue. Furthermore, access to our lab is strictly regulated, and it is extremely difficult for unauthorized individuals to access areas where E. coli is being stored or grown.<br />
</p><br />
<br />
<p style="color:black"><br />
<u><br />
Environmental safety<br />
</u><br />
</p><br />
<p style="color:black"><br />
&nbsp; &nbsp; The strains of bacteria that we use in our experiments pose little risk to the environment. This is mainly in part because these strains of bacteria require conditions that would be unlikely to be found outside of the laboratory.<br />
</p></div><br />
<div class="bigbox"><br />
<b class="blue"><br />
2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes,<br />
<ul class="grey"><br />
<li>Did you document these issues in the Registry?</li><br />
<li>How did you manage to handle the safety issue?</li><br />
<li>How could other teams learn from your experience?</li><br />
</ul><br />
</b><br />
<p style="color:black"><br />
&nbsp; &nbsp; While most of our devices and BioBrick parts do not pose any significant safety issues that have not already been addressed, we are planning to biobrick a cytotoxic protein known as clyA. ClyA variants have been used by iGEM teams in the past, however we were unable to determine what precautions they took when using the protein. We also evaluated literature where researchers used clyA, and again, could not find specific safety recommendations. Due to the lack of specific information, we performed all experiments involving clyA under BSL-2 precautions, which was the highest rating available to us. From our experience, we have found that the best resource when dealing with agents of unknown or unclear toxicity, such as clyA, is through the department responsible for laboratory safety at whichever institution the research is being performed. We plan to include the recommendations of our Biosafety Committee and our experiences with clyA in the Registry once our biobrick is submitted.<br />
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3. Is there a local biosafety group, committee, or review board at your institution?<br />
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<li>If yes, what does your local biosafety group think about your project?</li><br />
<li>If no, which specific biosafety rules or guidelines do you have to consider in your country?</li><br />
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&nbsp; &nbsp; As one of the world's foremost research institutions, the University of Pennsylvania has an extensive biological <a style="color:blue" href="http://www.ehrs.upenn.edu/programs/bio/bsm/">safety manual</a> that we abide by. Through these rules we have been approved for recombinant DNA lab work under a BSL-2 degsignation from the University EHRS after review. All members of our lab have completed a basic laboratory safety and biosafety program, as well as further training from postdocs and more experienced lab members. Currently our lab falls under the full BSL-2 designation for laboratories, which allows us to perform most of our work. Our mentor, Dr. Sarkar, has generously provided us with additional BSL-2 lab space that we can use to perform some of our other experiments. The BSL ratings and other biosafety regulations can be found <a href="http://www.ehrs.upenn.edu/programs/bio/bsm/principles.html" style="color:blue">here</a><br />
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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?<br />
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&nbsp; &nbsp; The current safeguards that most research institutions are designed primarily to protect the researcher from exposure to organisms, DNA, and chemicals that may cause harm. However, there is still room for improvement when considering the control of recombinant DNA (rDNA). For example, while unlikely, there is a possibility that a plasmid, perhaps containing a virulence factor, toxin, or antibiotic resistance gene could be transferred from a laboratory strain of bacteria to a more hardy strain without the knowledge of the researcher (horizontal gene transfer). Therefore, it may be useful for future iGEM teams to investigate ways that plasmids can be designed to minimize this risk. <br />
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We sought to create a system in which iGEM teams and labs can display any protein of their choosing on the surface of E. coli. We engineered a novel Suface Display BioBrick (BBa K811004) using the N and C terminal domains of the Ice Nucleation Protein (INPNC), which allows iGEM teams to fuse any desired protein to INPNC using a simple ligation protocol with BamHI and PstI restriction sites.</p><br />
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As a preliminary proof of concept for our INPNC-enabled system, we displayed the red fluorescent protein mCherry on the outer membrane of E. coli BL21. After sonication and centrifugation of induced cells, almost all of the mCherry was localized in the membrane fraction when fused to INPNC, whereas in the control Intein-mCherry fusion (which exhibits cytoplasmic localization), all of the mCherry was contained in the lysate (Figure 1).</p><br />
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<b>Figure 1</b></div>Figure 1: Surface display of mCherry using INPNC system. INPNC-mCherry and Intein-mCherry fusions were expressed in E. coli BL21 in the pET26b expression vector and Wood-Intein expression plasmid, respectively. When fused to INPNC, almost all mCherry was localized in the membrane fraction after sonication and centrifugation, while in the case of Intein-mCherry, all mCherry was localized in the cytoplasmic lysate.</div><br />
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<div style="text-align:center;font-size:34px;color:white;"><b>Ethical and Perceptual Issues<br><br>With Synthetic Bacterial Therapeutics</b></div><br><br />
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<p style="color:black;text-indent:30px;">In order for synthetic biology to make valuable advances in medicine and biotechnology, progress must also be made on the bioethics front. Scientific progress solely for the sake of scientific progress is invaluable for increasing human knowledge, but as engineers, we must strive to make useful systems with the ultimate possibility of improving some aspect of life. This means that our engineered systems must comply with ethical and social norms. Although the development of new technologies in the laboratory is relatively independent of public opinion, the acceptance and integration of new technologies is largely dependent on public perception; a technology is useful only if it is used.</p></div><br />
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<b><div class="name" align="center">Scientific Risks</div></b><br><br />
<p style="color:black;text-indent:30px;"><br />
One of the biggest ethical concerns surrounding synthetic biology and genetically engineered bacteria centers on risk. Two major components of risk are biosafety, the impact of engineered organisms of human health and the environment and biosecurity, the potential for these engineered organisms to be used maliciously [1].<br />
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<b><div class="name" align="center">Biosafety</div></b><br><br />
<p style="color:black;text-indent:30px;">Almost all emerging biotechnologies carry an inherent risk to biosafety, especially at early and uncertain stages of development. This is especially true for synthetically engineered organisms. Because the field is so nascent, many of the developments by synthetic biologists are cutting edge. Biologists are still studying and discovering new information about the basic rules governing biology. Many of the gene networks and biological mechanisms used by synthetic biologists are still being studied, with fundamental advancements being made every day. With information constantly changing as well as the inherently nondeterministic behavior of many biological systems, it's very difficult to rationally design engineered organisms that act exactly as anticipated. The Registry of Standard Biological Parts attempts to address the issue of uncertainty by classifying and characterizing modular biological components. However, biology is not modular; different proteins and genes interact with each other in still unknown ways. While piece by piece assembly works extremely well for other forms of engineering (such as electrical or mechanical), this is because engineers designed all of these components from scratch and have a very good grasp on how each part works and how these parts interact with each other. Synthetic biologists are not yet at a point where they can build components completely from scratch that act exactly as anticipated; the core components (genes) are usually isolated from nature. While fusion-proteins can be designed using existing proteins, synthetic biologists cannot reliably synthesize a DNA sequence for a specific function. When building synthetic systems using a mix of natural parts, it's difficult to predict with complete accuracy how the system will react in varying conditions and with the surrounding environment or with the human body [2]. An organism engineered to produce a therapeutic and deliver it to a site in the body could unknowingly also be producing a toxin or other harmful metabolite and releasing it into the bloodstream, an outcome that may not be evident until it has an opportunity to cause significant harm.</p><br />
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<p style="color:black;text-indent:30px;">Another biosafety hazard involves the risk of contamination. How will synthetic biologists ensure that any microorganism they engineer will stay where they want it to [3]. Bacteria and microorganisms are extremely mobile, able to travel between places through the very air we breathe. Pharmaceutical contamination of drinking water is already raising concerns [4]. Contamination by engineered microorganisms would be worse still, as these organisms have the ability to replicate and evolve and spread in ways that are difficult to anticipate. They may evolve to fill new niches or compete against natural organisms, altering ecosystems and biodiversity in unpredictable ways. Genetic pollution may also occur through horizontal gene transfer [2], giving natural organisms unexpected and potentially undesirable capabilities. Microorganisms are able exchange DNA with each other or uptake DNA from the environment. Leaked engineered bacteria could transfer resistance genes across species, similar to the StarLink corn controversy that surrounded genetically modified corn in 2000-2001. Aventis created a genetically modified corn called StarLink with Bacillus thuringiensis (Bt) derived insect resistance protein called Cry9c. Corn including this protein was restricted by the EPA to animal feed only due to potential for allergenicity, but traces of the Cry9c protein were found in human corn products due to genetic pollution of human feed, resulting in food recalls by the FDA and disruption of the food supply [5]. A similar gene transfer could occur in microbes; however, due to a shorter replication time, the suspect gene would spread much more quickly and a "bacteria recall" would be impossible to implement.</p><br />
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<b><div class="name" align="center">Biosecurity</div></b><br><br />
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<p style="color:black;text-indent:30px;">While synthetic biology has the potential to greatly improve human life, it is also capable of producing harmful microorganisms, either by accident (discussed above) or on purpose. Synthetic biology could open the door for a new type of bioterrorist – one that could rationally design microbiological weapons for use on the public. While DNA sequences and research publications may be readily available, construction of an actual organism capable of acting as a biological weapon (for example, by reconstituting a virus) is both difficult and expensive. Though it is still a possibility and needs to be taken as a serious issue, the equipment necessary to synthesize full viral or bacterial genomes is expensive, furthermore, facilities that offer these services screen synthesis orders to ensure that the DNA being synthesized poses no threat to public safety. Lastly as the DNA sequence alone is not enough to create a bio-weapon, the synthesis of potential harmful DNA is not sufficient to pose a threat to public safety by itself [6].</p><br />
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<b><div class="name" align="center">Intellectual Property and Ownership</div></b><br><br />
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<p style="color:black;text-indent:30px;">Another important ethical issue is the relationship between synthetic biology, intellectual property, and ownership. Should someone be allowed to patent a gene? What about a synthetic network of genes that perform a specific function? Should these systems be patentable or copyrightable? Or should they be neither and left to the public domain? While genes are currently patentable under the same law that makes drugs patentable, there is ongoing debate and controversy as to whether this law, should change as technological progress continues to redefine the assumptions made by legislators and regulatory agencies [7]. Broad patents on entire genes can slow down the growth and stifle innovation in the field, preventing the production of potentially useful systems. Early entrants into synthetic biology are attempting to patent basic genetic components to get ahead of their competition. Copywriting genetic code is not clear cut either, as copyright law generally requires expressive choice, which is limited by the 4 DNA base pairs.</p><br />
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<p style="color:black;text-indent:30px;">Putting the legal arguments aside, enforcing ownership of a gene or genetic system will be near impossible. Interested scientists can simply find the gene again in nature, or even in the engineered microorganism, and culture it themselves. Self-replicating bacteria that are distributed to the public and produce a therapeutic of choice could allow anyone interest to culture the organism themselves and have a potentially limitless supply of the drug. Simple PCR could allow direct access to the DNA "source code" for your own manipulations. This is much different than the current access to biologics or pharmaceuticals where only the final product, and not the means to produce for the product, is readily available. IP law will have to be very different to protect companies and provide incentives for the development of new biotechnologies while still promoting innovation and preventing the use of the patent system as a tool to monopolize and stifle competition.</p><br />
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<b><div class="name" align="center">Moral Issues</div></b><br><br />
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<p style="color:black;text-indent:30px;">The use of bacterial-based therapeutics may open some moral discussion on humanity and augmentation. With standard drug-based therapies, the only way to make the effect of the drug "permanent" would be to constantly re-administer doses. With bacteria therapeutics, it would be possible for the microbe to anchor in the body and produce a constant supply of a molecule. The passiveness of the therapy may bring up discussion about augmentation. By injecting these bacteria into the body, the person becomes better than they were previously, and this could theoretically be engineered to be permanent. Once this happens, you have changed the person, and philosophical discussions on the limits of man and the definition of human will most likely have to occur discussing the morals of altering the person. With socioeconomic factors in play, bacterial augmentations may widen the gap between the haves and have-nots. While the possibility of this happening would be many years into the future, it is still something that should be kept in mind, especially when thinking about public perception of synthetic biology.</p><br />
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<b><div class="name" align="center">Solutions</div></b><br><br />
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<p style="color:black;text-indent:30px;">The solutions to successfully integrate synthetic biology into society lie within the domains of regulation and control. Synthetic biology has the potential to do great good for humanity; it also has the potential to do great harm, whether intentionally or not. Therefore, measures must be put in place to ensure that the science is properly regulated and that synthetic organisms or systems are properly controlled.</p><br />
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<p style="color:black;text-indent:30px;"><br />
To combat the issues of biosafety and biosecurity, controls need to be placed on the organisms themselves. "Kill switches" that cause the bacteria to die under anything other than ideal conditions are a popular method of controlling synthetically modified organisms. Modifying the lifespan of the bacteria finite will lower the risk of accidental contamination or genetic pollution. Additionally, regulation such as the FDA clinical trial process for drugs and biologics will need to be developed for engineered bacterial therapeutics. Scientists developing these treatments must be held to high standards, and should provide extensive research demonstrating not only that their microbe works, but that it doesn't react in unexpected ways or produce unwanted byproducts in various conditions.</p><br />
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<p style="color:black;text-indent:30px;"><br />
Additional regulation must be enacted to ensure accountability and transparency among synthetic biologists. There should be accountability among labs. This may be difficult with the advent of the DIY "biohacker" cloning genes in their own private labs, but safety is of the utmost importance. Transparency and accountability helps prevent accidental leakage of microorganisms into the environment as well as careless or reckless science by unqualified people.</p><br />
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<p style="color:black;text-indent:30px;"><br />
The Presidential Commission on the Study of Bioethical Issues outlined 18 recommendations that they believe the government and synthetic biologists should take as the science moves forward [6]. All of the recommendations apply to engineered bacterial therapeutics. To see the success of these microorganisms and to allow synthetic biology to reach its full potential, scientists and engineers will have to work hand in hand with businessmen and policy makers to support innovation, evaluate risks, and promote a regulated and transparent industry to a well educated public.</p><br />
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<b><div class="name" align="center">Hurdles in Public Perceptions of Bacterial Therapeutics</div></b><br><br />
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<p style="color:black;text-indent:30px;"><br />
Even if an engineered bacterial therapeutic is developed, shown to be efficacious and safe, approved by relevant regulatory agencies, and launched, it will face hurdles in public and governmental perception. There is a reason why food products supplemented with bacterial cultures have been branded "probiotics"; there are clear negative associations with ingesting foreign bacteria. Unfortunately for projects like ours, there are also further negative associations with genetic engineering and synthetic biology.</p><br />
<p style="color:black;text-indent:30px;"><br />
Negative public opinion or low awareness can greatly hinder the progress of new technologies. Most notably, progress in stem cell research has been slowed by its initial negative public perception, which resulted in laws which outlawed or severely restricted this research in several countries. Although public opinion has recovered and 58% of the US population now approves of embryonic stem cell research [8], there is a long lag between changes in public opinion and changes in legislation, and progress has remained slow. To prevent synthetic bacterial therapeutics from following the same path, we must consider the potential public perception hurdles specific to synthetic biology and take action to minimize their impact.</p><br />
<p style="color:black;text-indent:30px;"><br />
Public acceptance of synthetic bacterial therapeutics is closely tied to public perception of synthetic biology in general. Since synthetic biology is still an emerging field, awareness is generally low but is increasing rapidly. According to a 2009 study by Hart Research Associates, roughly 48% of adults have never heard anything about synthetic biology, while 28% have heard "a little" about it, 22% have heard something about the field and 5% have heard a lot about the field [9]. Importantly, only 18% believed that the benefits of synthetic biology will outweigh the risks. Perceptions have since been influenced by the recent work of Venter, et al. in which they engineered bacteria from a "synthetic" chromosome [10]. This research propelled synthetic biology to the forefront of national discourse for the first time in 2010, resulting in widespread shifts in perception about synthetic biology and an increase in awareness. Discussions emerged about whether synthetic biologists were "playing God," which has had a negative impact on public opinion of the synthetic biology because this type of discourse has not been accompanied by public education on the reality of the field [11]. In addition, the distinction between genetic engineering and synthetic biology is often not well understood by the public, and the negative perceptions of genetically modified organisms (roughly half of the US population opposes GMOs [12]) can thus spill over to synthetic biology. Given this current opinion landscape, it is reasonable to assume that even if a synthetic bacterial therapeutic was shown to be safe and effective, it may encounter public resistance. The development of engineered bacterial therapeutics may also be hindered by an eventual decline in public interest in the field and parallel decreases in public and private funding. Since government research funding is largely dependent on public priorities, high interest and awareness of a field is generally beneficial to researchers in that field. Synthetic biology has recently been enjoying a rapid rise in awareness, which has been accompanied by a spike in research funding [13]. Some of this funding has already resulted in the development of novel proof-of-concept synthetic bacterial therapeutics [14,15] which may develop into new treatments with time. However, like many other fields before it (most notably nanotechnology), synthetic biology is likely to experience a drop in public interest in the near future.</p><br />
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<b><div class="name" align="center">Beating the Hype</div></b><br><br />
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<p style="color:black;text-indent:30px;"><br />
The Gartner Hype Cycle [16] is a model which describes the public awareness of emerging technologies. It predicts that in the early onset of a new technology ("technology trigger"), expectations of its potential become inflated and the public gains considerable interest in it. This has arguably been occurring recently with synthetic biology, as evidenced by widespread claims that artificial life has been created and that the field can create designer organisms from scratch. These claims have greatly increased interest in the field, but in the near future the public will have to come to the realization that synthetic biology has limitations and is very far from actualizing many of the goals which have been presented to the public [11]. At this point of disillusionment, public interest will decrease, most likely accompanied by a decrease in research funding. In the case of nanotechnology, the initial excitement of the field throughout the 2000s has been subsiding, and federal funding for the field is also being decreased [17]. If synthetic biology follows this same path (Fig. 1), difficulties may arise in the development of engineered bacterial therapeutics. The very early stage of this type of research, combined with the long lead times for preclinical development and approval of therapeutics, and the fact that no such therapy has previously been approved will result in a very long timescale for the introduction of engineered bacterial therapeutics. This means that both near-term and long-term decreases in research funding for synthetic biology could significantly hinder their development.</p><br />
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<p style="color:black;text-indent:30px;"><br />
It is clear that throughout all stages of research, engineered bacterial therapeutics will encounter hurdles in public perception including mixed opinions of synthetic biology, false impressions of the potential and dangers of the field, and potential decreases in interest and funding for the field. However, these hurdles can be overcome if several measures are taken by synthetic biologists (including iGEM teams), the lay public, the government and the media. Firstly, when iGEM teams, synthetic biologists, and the media describe advances in synthetic biology, we must strive for informational accuracy in our portrayals of the science. Phrases such as "creating life" and "playing god" detract from public opinion of the field. In response to this, the government has moved in the right direction by calling for an independent organization to fact-check the many claims made about synthetic biology which are disseminated to the public4. In parallel to such a strategy, improving public scientific literacy and education would decrease the likelihood that they accept such claims as truths. iGEM teams have contributed positively to this by engaging in outreach campaigns to the lay public, and the existence iGEM itself has greatly increased synthetic biology awareness and education among youth.</p><br />
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<p style="color:black;text-indent:30px;"><br />
If the public is well-educated about synthetic biology and understands its immense potential in medicine, we believe that synthetic bacterial therapeutics will overcome perception and funding hurdles in the near future and eventually treat diseases in ways which were not possible before.</p><br />
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<b><div class="name" align="center">FDA Regulatory Barriers to Synthetic Bacterial Therapeutics</div></b><br><br />
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<p style="color:black;text-indent:30px;"> Many iGEM teams produce projects that they hope will one day be applied to human health, or in some cases, are actually designed to be consumed by human beings. Any project that has consumption or medical use as its ultimate goal would undergo scrutiny from a wide array of regulatory agencies. This process can take a great deal of time and money, and is a major consideration for any biotechnology startup, including ones that may arise as a result of an iGEM project. Here, we focus specifically on the US regulatory agencies, but many regulatory practices in other regions follow similar guidelines.</p><br />
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<p style="color:black;text-indent:30px;"><br />
The Food and Drug Administration (FDA) is the primary regulatory agency for medical devices and pharmaceutical compounds. Historically, the agency has guided and reviewed clinical trials for medications, as well as outlined standards for the testing and approval of medical devices. The rapid development of the biotechnology industry has also prompted the FDA to make further distinctions between "chemical small molecule entities," (SME) and "biologics," which are defined as "any virus, therapeutic serum, toxin, antitoxin or analogous product applicable to the prevention, treatment or cure of diseases or injuries of man." This definition has since been applied to products of the biotechnology industry, including future products based on the work of the 2012 Penn iGEM team. Essentially, the FDA makes a distinction between products that are manufactured in a well characterized fashion, such as traditional pharmaceuticals and recently some recombinant proteins, and products that are produced in processes which are more variable, such as the growth of viruses in bioreactors [18].</p><br />
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<p style="color:black;text-indent:30px;"> The FDA is divided into two large divisions, the Center for Drug Evaluation and Research (CDER), which is responsible for evaluating SME drugs, and the Center for Biologics Evaluation and Research (CBER), which is responsible for evaluating biologics. It is likely that any bacterial-based therapeutics, such as the ones developed by the 2012 Penn iGEM team would most likely fall under the jurisdiction of CBER.</p><br />
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<p style="color:black;text-indent:30px;"> Under such regulations, a bacterial-based therapeutic would be required to undergo the biologic licence application process (BLA). During this process, the FDA would review not only the safety and efficacy of a biologic, but also the process by which a biologic is produced. SME drugs are often produced through established industrial processes, and are therefore biologics are extremely sensitive to small variations during the production process, and are therefore more strictly regulated. The FDA BLA process can be divided into five distinct stages [19].</p><br />
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<p style="color:black;text-indent:30px;"> The first stage is the "Filing Determination & Review Planning Stage," where reviewers from the FDA determine if the BLA meets the minimum standards that the FDA has set for filing an BLA, the amount of review required for the specific needs of the BLA, and the primary areas of the BLA that reviewers must focus on. FDA officials convene to produce a timeline for the rest of the review process. This process can be considered a planning stage, where all BLAs that meet the FDA requirements advance to the next stage [19].</p><br />
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<p style="color:black;text-indent:30px;"> The second stage is known as the "Review Phase," where FDA officials not only review the merits of the application itself, but also distribute the application to outside investigators, who can provide their own evaluations of the BLA. During this process, FDA officials are in constant contact with the BLA applicant, who may be asked to provide additional information depending on the requirements of the reviewers [19].</p><br />
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<p style="color:black;text-indent:30px;"> The third stage is known as the "Advisory Committee Meeting Phase." This stage occurs when "the clinical study design used novel clinical or surrogate endpoints, the application raises significant issues on the safety and/or effectiveness of the drug or biologic, or the application raises significant public health questions on the role of the drug or biologic in the diagnosis, cure, mitigation, treatment, or prevention of a disease". In the case of bacterial therapeutics, due to lack of precedent, any BLA based on such technology would most likely undergo this third step [19].</p><br />
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<p style="color:black;text-indent:30px;"> The fourth stage is known as the "Action Phase." During this phase, the FDA officials, based on the information obtained during the "Review Phase," will outline a series of requirement for the final therapeutic product. In this phase, specific requirements for the labeling of the product, as well as the conclusions reached by the FDA officials is summarized [19].</p><br />
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<p style="color:black;text-indent:30px;"> The final stage of the BLA approval process is known as the "Post-Action Phase," where FDA officials analyze their performance during previous BLA evaluation stages, and improve upon them for future BLA applications [19].</p><br />
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<p style="color:black;text-indent:30px;"> The BLA approval process is time consuming and can discourage the commercialization of may biologics. However, this process also has several advantages. Firstly, while SMEs are much easier to approve, they also allow for Abbreviated New Drug Applications (ANDAs). The ANDA process allows for generic drug manufacturers to produce generic versions of SMEs after five years, a factor that significanly reduces the incentive for applicants to submit an appliation for a SME. However, no ANDA analog exists for BLAs. Therefore, if an BLA is approved, it would be extremely difficult for other companies to produce "generic" versions of the biologic, creating a larger incentive for applicants to undergo the BLA approval process.</p><br />
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<b><div class="name" align="center">References</div></b><br><br />
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[1] Jain, A., Bhatia, P. & Chugh, A. Microbial synthetic biology for human therapeutics. Systems and Synthetic Biology (2012).doi:10.1007/s11693-012-9092-0<br><br><br />
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[2] Dana, G. V., Kuiken, T., Rejeski, D. & Snow, A. A. Four steps to avoid a Assess the ecological risks of synthetic microbes before they escape the lab ,. 2012 (2012).<br><br><br />
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[3] de S Cameron, N. M. & Caplan, A. Our synthetic future. Nature biotechnology 27, 1103–5 (2009).<br><br><br />
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[4] Bruce, G. M., Pleus, R. C. & Snyder, S. a Toxicological relevance of pharmaceuticals in drinking water. Environmental science & technology 44, 5619–26 (2010).<br><br><br />
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[5] Bucchini, L. & Goldman, L. R. Starlink corn: a risk analysis. Environmental health perspectives 110, 5–13 (2002).<br><br><br />
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[6] Commission, P. & Issues, B. New Directions: The Ethics of Synthetic Biology and Emerging Technologies. (2010).<br><br><br />
<br />
[7] Eisenberg, R. S. Why the gene patenting controversy persists. Academic medicine : journal of the Association of American Medical Colleges 77, 1381–7 (2002).<br />
<br />
[8] Roberts, J. (2, August 2010). Poll: Stem cell use gains support. Retrieved from http://www.cbsnews.com/2100-500160_162-697546.html<br><br><br />
<br />
[9] (2009). Nanotechnology, synthetic biology, & public opinion. Washington, DC: Hart Research Associates.<br><br><br />
<br />
[10] Gibson, D. G. et al. Creation of a bacterial cell controlled by a chemically synthesized genome. Science (New York, N.Y.) 329, 52–6 (2010).<br />
<br />
[11] Presidential Commission for the Study of Bioethical Issues, (2010). New directions: The ethics of synthetic biology and emerging technologies. Washington, DC: Hart Research Associates.<br><br><br />
<br />
[12] (2006). Review of public opinion research. Washington, DC: The Mellman Group.<br><br><br />
<br />
[13] (2010). Trends in synthetic biology research funding in the united states and europe. Washington, DC: Woodrow Wilson International Center for Scholars.<br><br><br />
<br />
[14] Chen, Y. Y. & Smolke, C. D. From DNA to targeted therapeutics: bringing synthetic biology to the clinic. Science translational medicine 3, 106ps42 (2011).<br><br><br />
<br />
[15] Ruder, W. C., Lu, T. & Collins, J. J. Synthetic biology moving into the clinic. Science (New York, N.Y.) 333, 1248–52 (2011).<br><br><br />
<br />
[16] Linden, A. (2003). Understanding gartner's hype cycles. Conshohocken: Gartner.<br><br><br />
<br />
[17] Sargent, J. F. (2011). Federal research and development funding: Fy2011. Washington, DC: Congressional Research Service.<br><br><br />
<br />
[18] Kathleen R. Kelleher, FDA Approval of Generic Biologics: Finding a Regulatory Pathway,<br />
<br />
14 Mich. Telecomm. Tech. L. Rev. 245 (2007)available at http://www.mttlr.org/volfourteen/kelleher.pdf<br><br><br />
<br />
[19] Wan, Elysa, Jeffrey Kopacz, and Kathleen Williams. Biological Licensing v. Drug<br />
<br />
Approval Processes: Comparison & Consequences. N.d. Legal Brief. Massachusetts,<br />
<br />
Boston<br />
</div><br />
</html></div>Qiaophttp://2012.igem.org/Team:Penn/TargetingTeam:Penn/Targeting2012-10-26T22:14:21Z<p>Qiaop: </p>
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<b><div class="name" align="center">Testing the Displayed HER2 DARPin on Cancer Cells</div></b><br /><br />
<br />
<p style="color:black;text-indent:30px;"><br />
<br />
After verifying that the DARPin was displayed on the cell surface, we sought to test whether it could allow binding to HER2-overexpressing cancer cells. We were generously provided SKBR3 cells by the lab of Matthew Lazzara. These cells are derived from breast cancer cells and over-express HER2. We also obtained Human Embryonic Kidney (HEK) 293T cells, which express a low amount of HER2, to act as controls.</p><br />
<br />
<p style="color:black;text-indent:30px;"><br />
<br />
To verify that our SKBR3 cells overexpressed HER2, we immunostained for HER2 (primary anti-Neu Mouse Igg, Santa Cruz Biotechnology and secondary donkey anti-mouse Alexa-Fluor 647 conjugate, Jackson Immunoresearch) and visualized the cells on a confocal microscope. As expected, HER2 was overexpressed in SKBR3 cells (Figure 10).</p><br />
<br />
<div class="figs2"><br />
<div class="fignew"><div align="center"><img src="https://static.igem.org/mediawiki/2012/e/e0/SKBR3_%28%2BDAPI%29_%28%2BAB1%29_%28%2BAB2%29_630x.jpg" height="400"><br><br />
<b>Figure 10</b></div>Figure 10: SKBR3 cells over-express HER2 on their surface. Red indicates HER2 (Alexa-Fluor 647), Blue indicates DAPI.</div><br />
<br />
<div class="fignew"><div align="center"><img src="https://static.igem.org/mediawiki/2012/7/7a/HER2-Breast-Tissue.jpg" height="400"/><br><br />
<b>Figure 11</b></div>Figure 11: Images of breast cancer cells removed from human tissue reveal similar HER 2 expression to results obtained in Figure 10. </div></div><br />
<br />
<br />
<br />
<p style="color:black;text-indent:30px;">To assay whether our DARPin-displaying E. coli bound to SKBR3 cells preferentially, we conducted experiments in which our bacteria were co-incubated with SKBR3 or HEK293T cells. The DARPin-displaying bacteria were co-transformed with eGFP for easy visualization. These experiments are still in progress to optimize the co-incubation and gentamycin protection protocol (we designed this type of experiment ourselves since it has not been done before). However, preliminary results are exciting and show preferential binding to SKBR3 cells (Figure 12).</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/a/a6/Darpinbinding2.png" width="794" height="425" /></div><br />
<br />
<div class="fig11"><b>Figure 12</b>: SKBR3 cells overexpress HER2 and DARPin-displaying bacteria bind to their surface. Red indicates HER2 (Alexa-Fluor 647). Blue indicates DAPI. Green indicates eGFP. Bacteria were co-incubated at a Multiplicity of Infection of 200:1 and washed three times after co-incubation followed by a 50ug/mL gentamicin protection assay.</p></div><br />
<br />
<br />
<br />
In summary, we demonstrated surface display of DARPin H10-2-G3 for the first time and have preliminary evidence showing our DARPin-displaying bacteria can target cancer cells. We also developed a generalized surface display BioBrick for any iGEM team to use.<br /> </div></div><br />
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<b><div class="name" align="center"><br />
Displaying an Engineered Cancer Cell Binder on the Outer Membrane Surface<br />
</div></b><br /><br />
<p style="color:black;text-indent:30px;"><br />
Having demonstrated that our INPNC surface display system could localize a desired protein (mCherry) to the outer membrane surface, we sought to display the picomolar affinity, HER2 binding Designed Ankyrin Repeat Protein (DARPin) H10-2-G3 on the surface of our BL21 cells. This would allow for bacterial targeting to HER2 over-expressing cells, that could then be lysed in a spatially accurate manner using our light-activated ClyA expression system. Such surface display of a large antibody-mimetic protein is unprecedented, and assaying whether it had actually occurred would be difficult.</p><br />
<p style="color:black;text-indent:30px;"><br />
We constructed a C-terminal INPNC-DARPin fusion in the same manner as our INPNC-mCherry fusion, except we added a Human Influenza Aggregation (HA) tag to the N-terminal of DARPin to allow us to use antibodies to assay whether it had been localized to the membrane. After expression and 48-hour induction of INPNC-DARPin-HA in BL21 cells using the pET26b expression vector, we immunostained cells using an anti-HA, Alexa-fluor 647 conjugated antibody (Cell Signaling Technologies). Since antibodies are not permeable to the E. coli membrane, after washing the cells there should be signal only on those cells that have displayed the HA tag on their surface. We attempted to assay these stained cells with flow cytometry, but soon realized that we required much higher-resolution FACS sorting equipment than we had available to count bacteria.</p><br />
<p style="color:black;text-indent:30px;"><br />
We then took a small amount of HA immunostained bacteria and visualized them by confocal microscopy at high (630x) magnification using a 633 He-Ne laser. Using this immunostaining visualization method, we were able to demonstrate for the first time that DARPin H10-2-G3 had in fact been localized to the cell surface (Figures 2-9).</p><br />
<p style="color:black;text-indent:30px;"><br />
We first stained control bacteria expressing DARPin-HA without fusion to INPNC (Figures 2-3). The following images were taken of identical densities of bacteria as verified by phase contrast.</p><br />
<br />
<div class="figs2"><br />
<div class="fignew"><div align="center"><img src="https://static.igem.org/mediawiki/2012/2/2b/His-DARPin-HA_%28-IPTG%29_%28%2BAB%29.jpg" width="250" height="250"/><br><br />
<b>Figure 2</b></div>Figure 2: Uninduced pET26b-DARPin-HA (-IPTG) bacteria are not immunostained by anti-HA antibody. </div><br />
<br />
<br />
<div class="fignew"><div align="center"><img src="https://static.igem.org/mediawiki/2012/7/74/His-DARPin-HA_%28%2BIPTG%29_%28%2BAB%29.jpg" width="250" height="250"/><br><br />
<b>Figure 3</b></div>Figure 3: Induced pET26b-DARPin-HA (+IPTG) bacteria are not immunostained by anti-HA antibody.</div></div><br />
<br />
<p style="color:black;text-indent:30px;"><br />
Then, to determine whether INPNC could display an HA tag on the surface of E. coli, we stained bacteria expressing INPNC-HA and found that IPTG-induced bacteria exhibited strong red fluorescence when excited by the 633nm laser (Figures 4-5).<br />
</p><br />
<br />
<div class="figs2"><br />
<div class="fignew"><div align="center"><img src="https://static.igem.org/mediawiki/2012/d/d0/INPNC-HA_%28-IPTG%29_%28%2BAB%29.jpg" width="250" height="250"><br><br />
<b>Figure 4</b></div>Figure 4: Uninduced pET26b-INPNC-HA (-IPTG) bacteria are not immunostained by anti-HA antibody.</div><br />
<br />
<div class="fignew"><div align="center"><img src="https://static.igem.org/mediawiki/2012/6/6b/INPNC-HA_%2BIPTG.JPG" width="250" height="250"><br><br />
<b>Figure 5</b></div>Figure 5: Induced pET26b-INPNC-HA (+IPTG) bacteria are strongly immunostained by anti-HA antibody, indicating surface expression.</div></div><br />
<br />
<br />
<br />
<p style="color:black;text-indent:30px;"><br />
Finally, to determine whether we could display our HA-tagged DARPin on the surface of E. coli, we stained INPNC-DARPin-HA expressing bacteria and found strong immunofluorescence under the induced condition (Figures 6-7).<br />
</p><br />
<div class="figs2"><br />
<div class="fignew"><div align="center"><img src="https://static.igem.org/mediawiki/2012/4/46/INPNC-DARPin-HA_%28-IPTG%29_%28%2BAB%29.jpg" width="250" height="250"><br><br />
<b>Figure 6</b></div>Figure 6: Uninduced pET26b-INPNC-DARPin-HA (-IPTG) bacteria are not immunostained by anti-HA antibody</div><br />
<br />
<div class="fignew"><div align="center"><img src="https://static.igem.org/mediawiki/2012/5/58/INPNC-DARPin-HA_%2BIPTG_%2BAB.JPG" width="250" height="250"><br><br />
<b>Figure 7</b></div>Figure 7: Induced pET26b-INPNC-DARPin-HA (+IPTG) bacteria are strongly immunostained by anti-HA antibody, indicating surface expression.</div></div><br />
<br />
<br />
<p style="color:black;text-indent:30px;"><br />
We also repeated the experiment without the antibody to rule out any possibility of autofluore<div class="figs2">scence. As expected, no fluorescence was detected when INPNC-DARPin-HA bacteria were not stained with the anti-HA Alexa-Fluor 647 antibody (Figures 8-9).</p><br />
<div class="figs2"><br />
<div class="fignew"><div align="center"><img src="https://static.igem.org/mediawiki/2012/4/46/INPNC-DARPin-HA_%28-IPTG%29_%28%2BAB%29.jpg" width="250" height="250"><br><br />
<b>Figure 8</b></div>Figure 8: Uninduced pET26b-INPNC-DARPin-HA (-IPTG) (-AB) bacteria are not fluorescent. </div><br />
<br />
<div class="fignew"><div align="center"><img src="https://static.igem.org/mediawiki/2012/b/b3/INPNC-DARPin-HA_%28%2BIPTG%29_%28-AB%29.jpg " width="250" height="250"><br><br />
<b>Figure 9</b></div>Figure 9: Induced pET26b-INPNC-DARPin-HA (-IPTG) (-AB) bacteria are not fluorescent.</div></div><br />
<br />
<br />
<p style="color:black;text-indent:30px;"><br />
All controls performed as expected, and we are very confident that our DARPin has been displayed on the surface of E. coli.<br />
</p><br />
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<b><div class="name" align="center">Light-Dependent Lysis of Mammalian Cells by Bacteria</div></b><br /><br />
<br />
<p style="color:black;text-indent:30px;"><br />
We then wanted to prove that our pDawn-ClyA construct was able to lyse mammalian cells in a light-dependent manner. To assess this, we plated BL21 bacteria transformed with pDawn-ClyA or pDawn-mCherry on Columbia Agar plates supplemented with 5% sheep blood (BD). These plates are used to qualitatively detect hemolytic activity in bacteria by visually confirming lysis through a color change in the media as the blood cells are lysed. After plating the bacteria, cultures were grown in non-inducing conditions at 37°C until visible colonies were present (~12 hours). Plates were then grown at 25°C under either inducing or non-inducing conditions for 24 hours and imaged. These results are visible in Figure 4.</p><br />
<br />
<div class="figs2"><br />
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pDawn-mCherry Dark</div></div><br />
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<br><br />
<div class="figs2"><br />
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pDawn-His-ClyA Dark</div></div><br />
<br />
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pDawn-His-ClyA Light</div></div></div><br />
<br />
<br />
<div class="fig"><br />
<div align="center"><b>Figure 4</b></div></div><br />
</div><br />
<div class="bigbox"><br />
<b><div class="name" align="center">Spatial Cell Lysis</div></b><br /><br />
<div class="figs2"><br />
<div class="fignew"><div align="center"><img src="https://static.igem.org/mediawiki/2012/7/76/Colony-Spatial-Lysis---Grey-BG.jpg " height="400"><br><br />
<b>Figure 5</b><br><br />
Figure 5: Colony Spatial Control.</div></div><br />
<br />
<div class="fignew"><div align="center"><img src="https://static.igem.org/mediawiki/2012/1/10/Penn-iGEM-Lysis---Grey-BG.jpg" height="400"><br><br />
<b>Figure 6</b><br><br />
Figure 6: Penn iGEM spatial control</div></div></div> </div><br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Verification of Expression of Cytolysin A (ClyA)</div></b><br /><br />
<div class="fig"><div align="center"><img src="http://partsregistry.org/wiki/images/3/3a/Gel_pic_pdawn.png"><br><br />
<br><br />
<b>Figure 7</b><br><br />
Figure 7: The production of clyA-his in BL21 in both bacteral lysate and culture medium</div></div> <br />
<br />
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<b><div class="name" align="center">pDawn and Nissle 1917</div></b><br /><br />
<br />
<p style="color:black;text-indent:30px;"><br />
In order to further develop our system for future in vivo therapeutic applications, we transformed Nissle 1917 with pDawn-mCherry to see if we could implement our system into a non-pathogenic strain of E. coli. We repeated our initial experiments and achieved light-dependent gene expression in Nissle 1917 for the first time ever. We are now hoping to clone in our pDawn-ClyA construct to show that Nissle 1917 is capable of light-dependent lysis of mammalian cells. Stay tuned!<br />
</p><br />
<br />
<div class="fig"><div align="center"><img src="https://static.igem.org/mediawiki/2012/7/72/Nissle-1917-pDawn-mCherry-10-1-2012.jpg" width="250" height="350"><br><br />
<br><br />
<b>Figure 8</b></div></div><br />
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<b><div class="name" align="center">YF1/FixJ Characterization</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;">After reading many papers to select an appropriate light-sensing system to use, we selected the YF1/FixJ blue light system. We had also considered the red light sensor Cph8 but ultimately decided on YF1/FixJ because of its high on/off ratio of gene expression and also because of its availability to us (we were fortunate enough to come across the YF1/FixJ system in the form of the pDawn plasmid from the Moglich lab in Germany).</p><br />
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<b><div class="name" align="center">YF1/FixJ System (pDawn)</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"> As shown below in Figure 1, the YF1/FixJ system works through a "repress the repressor" concept. Upon 480 nm blue light illumination, YF1 (a fusion of a LOV protein domain and a histidine kinase) phosphorylates a FixJ response regulator that activates the pFixK2 promoter. The activation of pFixK2, promotes expression of the cI repressor that, in turn, represses the lambda promoter pR. The net result is activation of the gene in the downstream MCS. </p><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2012/7/74/PDAWN.gif" width="500" height="300" /><br />
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<div style="text-align:center"><b>Figure 1</b><br /></div><br />
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<br />
<br />
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<div class="bigbox"><br />
<b><div class="name" align="center">YF1/FixJ (pDawn) Objectives </div></b><br />
<br><br />
To characterize our pDawn gene expression system, we showed the following:<br />
<ol><br />
<li> pDawn allows for light-dependent gene expression in bacteria.<br />
<li> pDawn allows for light-dependent lysis of mammalian cells by bacteria.<br />
</ol><br />
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<br />
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<br />
<b><div class="name" align="center">Light-Dependent Gene Expression in Bacteria</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;">We tested for light-dependent gene expression by cloning an mCherry reporter protein into the multiple cloning site of the pDawn system. First, we tested for the on-off ratio by growing cultures of BL21-pDawn-mCherry in both inducing and non-inducing conditions for 22 hours. After spinning down the cultures in a centrifuge, we were able to visually confirm the expression of mCherry due to the bacterial pellet grown in inducing conditions to be colored red, while the other pellet had no color (Figure 2).</p><br />
<br />
<div align="center"><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/f/f4/Clark_Park_4.JPG" width="180" height="300"><br />
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<br><br />
<div style="text-align:center"><b>Figure 2</b><br /></div><br />
</div><br />
<br />
<br />
<br />
<div class="bigbox"><br />
<b><div class="name" align="center">Characterizing Time-Dependent Gene Expression</div></b><br />
<br><br />
<p style="color:black;text-indent:30px;"><br />
We then characterized the induction kinetics of the pDawn system through an mCherry expression time course. We induced cultures of BL21 pDawn-mCherry for 0 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, and 22 hours in a 37°C incubator shaking at 225 rpm, and then transferred them into a dark incubator under the same condition for the remaining growth period. After 24 hours, mCherry fluorescence was read on a Tecan Infinite m200 plate reader and normalized by OD. The cultures were then spun down in a centrifuge. These results can be seen in Figure 3. </p><br />
<br><br />
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<div style="text-align:center"><b>Figure 3</b><br /></div><br />
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<b><div class="name" align="center">A Novel Therapeutic Platform</div></b><br />
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<p style="color:black;text-indent:30px;">What if you could combine spatial targeting and cellular targeting into the same therapeutic? This idea is unprecedented but would allow for precise targeting of specific cells within a specific area, leaving healthy tissue intact and keeping side effects to a minimum.</p><br />
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<p style="color:black;text-indent:30px;">The 2012 Penn iGEM team has engineered a novel platform for targeted therapeutics that employs simultaneous spatial and cellular targeting. We have achieved spatial (and temporal) targeting with a blue light-switchable transgene expression system and cellular targeting through display of an antibody-mimetic protein on the surface of E. coli for the first time.</p><br />
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<p style="color:black;text-indent:30px;">As a proof of concept, we applied our system to the treatment of cancer, a disease in which spatial and cellular targeting are of utmost importance. We displayed a high-affinity antibody-mimetic protein that targets Human Epidermal Growth Factor Receptor 2 (HER2), a protein commonly overexpressed in cancer cells, especially in breac cancer tumors. We combined this cellular targeting with a light-activated cytotoxic protein delivery system to successfully target and kill breast cancer cells.</p><br />
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<b><div class="name" align="center">Human Practices</div></b><br />
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<p style="color:black;text-indent:30px;">Upon conception of this project, we realized that although hundreds of academic research projects and iGEM projects have been conducted in the realm of Health and Medicine, almost no engineered bacterial therapeutics have been brought to the clinic. We analyzed the hurdles and road ahead for bacterial synthetic biology-enabled therapeutics, compiling a thorough report with specific actions that iGEM teams in Health/Medicine can take to make their therapies more clinically tractable. This project directly informed our wet lab work, leading us to port our therapeutic system into a non-pathogenic, probiotic bacterial strain which is already used in human therapies today.</p><br />
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<p style="color:black;text-indent:30px;">We hope our targeted therapeutic platform will allow other scientists and iGEM teams to target any cells they choose. In the near term, we are planning to test our cancer cell targeting/killing bacterial system in a mouse model and make a real impact on cancer research and therapy.</p><br />
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<b><div class="name" align="center">Motivation</div></b><br /><br />
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Medicine is a challenge of <b>optimization.</b> When treating a disease, doctors and physicians want to <b>maximize</b> the on-target, beneficial effects of a therapy while <b>minimizing</b> the off-target or side effects. There are two main optimization parameters that doctors face when applying current medical therapies: specificity and dosage control. <br />
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<b><div class="name" align="center">Specificity</div></b><br />
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Current therapies are usually able to target spatially (in a particular region of the body) or cellularly (targeting to a specific cell type). However, these types of targeting still lead to high nonspecific effects and are limited in their approach. For example, radiation therapy is able to spatially target and kill tumor cells, but this works only when the cancer is <b>highly localized</b> and when the tumor area is well defined. Chemotherapy and monoclonal antibody therapies are able to target by cell type, but they target even healthy cells that are of that type. This is why patients undergoing chemotherapy exhibit horrible side effects: the treatment targets and <b>kills all rapidly dividing cells</b>, including hair and skin cells.<br />
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<b><div class="name" align="center">Dosage Control</div></b><br />
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<p style="color:black;text-indent:30px;">Precise dosage control is difficult in current medical therapies. Relying on passive diffusion makes dose precision hard to determine – of the dose injected into the body, how much of it actually reaches its target? Innovation in pharmacology therefore focuses on increasing the <b> therapeutic window </b> - the range of drug dose that is considered both effective and safe – so that doses may be increased if necessary. Besides being expensive in both cost and time, this shotgun approach toward therapeutics is inefficient. Essentially, it becomes a race – will the treatment kill the disease before it kills the patient?</p><br />
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<b><div class="name" align="center" style="font-size:20px;"><a href="https://2012.igem.org/Team:Penn/ProjectResults">How Can Synthetic Biology Improve Medicine?</a></div></b><br />
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