Team:UCSF/lily

From 2013.igem.org

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<h2>ABSTRACT</h2>
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    The human race is growing fast. The world population is projected to top 10 billion by 2050. One response to the food supply needs of an increasing world population is creating better crop yield through crop survival. Pathogens and pests can cause catastrophic crop loss. Currently there is no widely used synthetic biological mechanism to help plants fight off bacterial pathogens and pests, so the main way large-scale growers protect crops is by using pesticides. We have engineered two possible solutions to address this problem.
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    In our first project, we have incorporated catalytically dead CAS9 (dCAS9) in conjunction with synthetic gRNAs complementary to pathogenic bacterial genes into a plasmid that naturally conjugates into neighboring bacterial cells. The specificity of the gRNAs will potentially allow us to target specific populations of bacterial pathogens in soil while leaving positive bacterial populations unharmed.
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    Our second project will provide plants with a sugar-mediated synthetic circuit that boosts a plant’s natural immune system and pesticide-producing capabilities in response to different levels of sugar a plant naturally produces in response to pests. The repression capabilities of dCAS9 will be used to regulate the synthetic circuit promoters that sense high or low sugar levels, reducing metabolic strain.
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<h2>Special Thanks to our 2013 iGEM Team Sponsors!</h2>
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<li><a href='https://2012.igem.org/Team:Slovenia'><span>Home</span></a></li>
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<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li>
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    <li><a href="https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation"><table  onclick="window.location = 'https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation';" class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li>
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Implementation</span></a>
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  <ul>
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<li><a href='https://2012.igem.org/Team:Slovenia/Implementation'><span>Overview</span></a></li>
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<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li>
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<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li>
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    <li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ImplementationImpact';" class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li>
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Modeling</span></a>
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  <ul>
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<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li>
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    <li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingPK';" class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li>
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<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li>
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<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li>
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<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li>
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<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel';" class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li>
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    <li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations';" class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li>
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<li><a href='https://2012.igem.org/Team:Slovenia/Parts'><span>Parts</span></a></li>
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Notebook</span></a>
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<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li>
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    <li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/NotebookLablog';" class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li>
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<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li>
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Society</span></a>
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<li><a href='https://2012.igem.org/Team:Slovenia/Society'><span>Human practice</span></a></li>
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<li><a href='https://2012.igem.org/Team:Slovenia/SocietyScientists'><span>Scientists</span></a></li>
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<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedicalDoctors'><span>Physicians</span></a></li>
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<li><a href='https://2012.igem.org/Team:Slovenia/SocietyEthics'><span>Ethics, safety and regulations</span></a></li>
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<li><a href='https://2012.igem.org/Team:Slovenia/SocietyPatients'><span>Patients</span></a></li>
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<li><a href='https://2012.igem.org/Team:Slovenia/SocietyMedia'><span>Journalists and general public</span></a></li>
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<li><a href='https://2012.igem.org/Team:Slovenia/SocietyOutreach'><span>Outreach</span></a></li>
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<li><a href='https://2012.igem.org/Team:Slovenia/SocietyQuestionnaire'><span>Questionnaire</span></a></li>
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<li class='has-sub '><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team</span></a>
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<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li>
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<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li>
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<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table  onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TeamCollaborations';" class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li>
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<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li>
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<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li>
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<strong style="font-size:120%;">The Challenge</strong><br/>
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<p>Biological drugs such as hormones, enzymes, cytokines or antibodies are increasingly used to treat different diseases. Due to systemic administration, these drugs often have adverse effects. Additionally, the high cost of biopharmaceuticals imposes a heavy burden on health systems.</p>
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<p>We aimed to use the principles of synthetic biology to develop an advanced and safe method of <i>in situ</i> production of biological drugs to increase the quality of patients' lives. This system should increase compliance to therapy, decreas the number of required invasive drug administrations and enable advanced multistage therapy while local administration could reduce the side effects.  </p>
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<p>We addressed this challenge by implementing microencapsulated engineered mammalian cells that can be regulated from the outside to produce different therapeutics. This system should be effective, reliable, safe and potentially more cost-effective.</p>
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<a href="https://2012.igem.org/Team:Slovenia/TheSwitch"><img class="pic" src="https://static.igem.org/mediawiki/2012/0/0b/Svn12_homepage_1switch.png" width="300"  height="300" /></a>
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<strong>The switch</strong>
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<p> Switches are the basic regulatory elements through which the state of cells can be controlled. We <b>designed a new type of a universal bistable toggle switch for mammalian cells</b> based on designed DNA-binding proteins. Its properties enable simultaneous introduction of several orthogonal switches and construction of complex logic devices. We found that a <b>classic toggle switch topology is ineffective</b> if based on TAL effectors. Our modeling explaines that the reason for this is  noncooperative binding of monomeric TAL effectors to DNA. We solved this problem by introducing nonlinearity through the addition of the addition of <b>positive feedback loops</b> consisting of pairs of TAL activators and repressors competing for binding. This arrangement resulted, both theoretically and experimentally in mammalian cells, in functional switch that can be regulated by small molecule inducers. <a href="https://2012.igem.org/Team:Slovenia/TheSwitch">Read more...</a></p>
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<a href="https://2012.igem.org/Team:Slovenia/SafetyMechanisms"><img class="pic" src="https://static.igem.org/mediawiki/2012/a/a8/Svn12_homepage_2safety.png" width="300" height="300"/></a>
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<strong>Safety</strong>
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<p>In order to enable safe application of engineered cells in the microencapsulation based therapy we designed three safety mechanisms to <b>degrade the alginate capsules </b> at the end of the therapy, terminate the therapeutic cells by induction of apoptosis and to <b>tag any escaped cells for elimination</b> by the host's natural killer cells. <a href="https://2012.igem.org/Team:Slovenia/SafetyMechanisms">Read more...</a></p>
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<a href="https://2012.igem.org/Team:Slovenia/Implementation"><img class="pic" src="https://static.igem.org/mediawiki/2012/3/3d/Svn12_homepage_3implementation.png" width="300" height="300" /></a>
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<div class="ttip">
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<strong>Implementation</strong>
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<p>In <b>consultations with medical experts</b> we tailored our therapeutic devices based on implanted microencapsulated engineered cells to the treatments of hepatitis C and ischaemic heart disease by <i>in situ</i> <b>production of therapeutic protein effectors whose efficiency has already been demonstrated</b>. In agreement with our pharmacokinetic models, this strategy could <b>reduce side effects and improve efficiency of these therapies</b>. Switching between production of effectors with antiviral or anti-inflammatory and tissue regenerative effect could be regulated by administrating a small molecule inducer.
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  <a href="https://2012.igem.org/Team:Slovenia/Implementation">Read more...</a></p>
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<a href="https://2012.igem.org/Team:Slovenia/Modeling"><img class="pic" src="https://static.igem.org/mediawiki/2012/0/0a/Svn12_homepage_4modelling.png" width="300" height="300" /></a>
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<div class="ttip2">
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<strong>Modeling</strong>
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<p>Modeling was used to simulate and improve the properties of the switch and the pharmacokinetic distribution of drugs in the tissue, which is required for an effective therapy and decreased side effects. We introduced <b>improved methods of switch simulations</b> such as a quantitative parameter derivation and algorithmic/mixed simulation that can capture mixed regulator binding to operators. We also included into the wiki a server <b>for online switch simulation</b>. A pharmacokinetic model of the local delivery of therapeutics by microencapsulated cells predicted that this type of drug delivery should have reduced systemic side effects.</p><p><b>Exhaustive modeling</b> of the switch <b>demonstrated that the classic genetic toggle switch arrangement is not stable without cooperativity</b>, but it <b>confirmed functionality and improved robustness of the our switch design</b> with <b>two positive feedback loops. This topology does not require cooperativity since nonlinearity is introduced by the positive feedback loop. <a href="https://2012.igem.org/Team:Slovenia/Modeling">Read more...</a></b></p>
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<a href="https://2012.igem.org/Team:Slovenia/Society"><img class="pic" src="https://static.igem.org/mediawiki/2012/f/fa/Svn12_homepage_5society.png" width="300" height="300" /></a>
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<div class="ttip2">
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<strong>Society</strong>
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<p>Different aspects of general <b>medical applications of synthetic biology</b> and our project specifically <b>were discussed with a wide range of stakeholders, including medical professionals, patients</b>, experts on the law and ethics of GMO use and release, scientists, the media and the general public<b> that will all have to participate in a successful introduction of synthetic biology applications to clinical use</b>. We attempted to organize a <b>network of Slovenian high schools</b> to share the excitement of synthetic biology with <b>younger generations</b> and to demonstrate its application in <b>medicine and other fields</b>.  <a href="https://2012.igem.org/Team:Slovenia/Society">Read more...</a></p>
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<td class="splash">
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<a href="https://2012.igem.org/Team:Slovenia/Implementation"><img class="pic" src="https://static.igem.org/mediawiki/2012/d/d2/Svn12_homepage_6future.png" width="300" height="300" /></a>
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<div class="ttip2">
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<strong>Perspectives</strong>
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<ul style="padding-left:30px;">
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<li> We anticipate that designed DNA-binding element-based transcriptional factor logic will play a very important role in the development of synthetic biology,</li>
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<li>TAL-based switches allow simultaneous introduction of multiple switches to adopt multiple cellular states with numerous medical and other applications ,</li>
+
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<li><b>The safety</b> mechanisms, <b>including the inducible leak-free termination (prodrug), escape detection and capsule degradation components will allow implementation for different therapeutic purposes</b>,</li>
+
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<li>for an effective application <b>stable cell lines</b> containing integrated switches with selected therapeutic effectors and safety mechanisms will have to be established, most likely by the use of human artificial chromosomes,</li>
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<li>we plan to <b>initiate <i>in vivo</i> experiments</b> first <b>on ischaemia and wound healing</b>.</li>
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</ul>
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<p><a href="https://2012.igem.org/Team:Slovenia/Implementation">Read more...</a></p>
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<strong style="font-size:120%;">Abstract in plain english</strong><br/>
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<p>Biological drugs are being used ever more often as advanced drugs for the treatment of numerous diseases, due to their more specific mode of action. In current therapies the biological drugs are usually distributed more or less throughout the whole body, although each function should often be restricted to a specific organ or tissue. This can cause serious side effects, requires larger dosages and consequently raises the price of therapy. Our solution to this problem was to implant cells producing biological drugs inside the very tissue where the drug is required. The drug producing cells are safely sealed inside microcapsules that prevent cells from spreading throughout the body and protect them from destruction by cells of the host immune system. We constructed a device that allows implanted cells to produce different types of drugs while switching between those production states can be controlled from the outside by a physician, depending on the stage of the disease. We designed our device specifically for the therapy of hepatitis C or heart attack. Against hepatitis C the engineered cells produce a protein with antiviral activity, whose biological activity we have tested and confirmed. After the state of the cells is switched, a protein that improves liver regeneration would be produced. For the therapy after a heart attack we designed cells to suppress local inflammation and promote formation of new blood vessels only around the affected tissue. A physician may initiate self-destruction of the therapeutic cells and capsules by an outside stimulus when the therapy is complete or at any other given time. We believe our system to be safe, effective and applicable in the real world for the therapy of different types of diseases.</p>
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<strong style="font-size:120%;">Achievements in technical details</strong><br/>
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<ul style="padding-left: 30px;">
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<li>We present the first experimental implementation of a bistable toggle switch in mammalian cells based on noncooperative DNA-binding proteins as well as the first demonstration of a bistable switch based on designed DNA-binding proteins,</li>
+
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<li>a bistable toggle switch based on orthogonal TAL-repressors and activators was designed and tested in mammalian cells,</li>
+
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<li>mathematical modeling demonstrated improved robustness of a switch based on a positive feedback loop with respect to leaky transcription,</li>
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<li>we introduced three safety mechanisms into microencapsulated mammalian cells:
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<ul class="circle" style="padding-left: 50px;">
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<li>a tag for escaped cells enabling elimination by natural killer cells,</li>
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<li>a secretory alginate lyase for degradation of alginate microcapsules,</li>
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<li>apoptosis of therapeutic cells initiated by the addition of a prodrug.</li>
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</ul>
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</li>
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<li>We introduced interferon alpha and hepatocyte growth factor as an effector pair for the therapy of hepatitis C to inactivate the virus followed by the promotion of liver regeneration, </li>
+
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<li>  we introduced IL-1 receptor antagonist (anakinra) and vascular endothelial growth factor/platelet-derived growth factor BB as effectors for therapy of ischaemia to suppress inflammation followed by the local promotion of angiogenesis,</li>
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<li>we deposited 89 BioBricks to the Registry and used most of them in functional devices,</li>
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<li>we improved an existing BioBrick.</li>
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<strong style="font-size:120%;font-color:gray;">Project sponsors</strong><br/>
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<a href="http://enfist.si/"><img width="100%" src="https://static.igem.org/mediawiki/2012/1/18/Svn12_sponsors_enfist.png"></img></a><br/><br/><br/>
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<a href="http://www.lek.si/"><img  width="80%" src="https://static.igem.org/mediawiki/2012/f/f1/Svn12_sponsors_lek.jpg"></a></img><br/><br/><br/>
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<a href="http://www.kemomed.si/"><img width="50%" src="https://static.igem.org/mediawiki/2012/d/da/Svn12_sponsors_kemomed.jpg" ></a></img><br/><br/><br/>
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<a href="http://www.roche.si/"><img width="50%" src="https://static.igem.org/mediawiki/2012/3/3b/Svn12_sponsors_roche.jpg"></a></img><br/><br/><br/>
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<a href="http://www.osir.si/"><img width="40%" src="https://static.igem.org/mediawiki/2012/7/75/Svn12_sponsors_osir.jpeg"></a></img><br/><br/><br/>
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<a href="http://www.adria.si/"><img width="40%" src="https://static.igem.org/mediawiki/2012/9/9f/Svn12_sponsors_adria.png"></a></img>
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<a href="http://www.ki.si/"><img width="100%" src="https://static.igem.org/mediawiki/2012/7/7e/Svn12_sponsors_ki.png" ></a></img><br/><br/><br/>
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<a href="http://www.fri.uni-lj.si/"><img width="80%" src="https://static.igem.org/mediawiki/2012/6/6b/Svn12_sponsors_fri.png" ></img></a><br/><br/><br/>
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<a href="http://www.mf.uni-lj.si/"><img width="60%" src="https://static.igem.org/mediawiki/2012/a/a1/Svn12_sponsors_mf.PNG"/></a><br/><br/><br/>
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<a href="http://www.krka.si/"><img width="50%" src="https://static.igem.org/mediawiki/2012/8/84/Svn12_sponsors_krka.png"></img></a><br/><br/><br/>
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Revision as of 23:25, 14 August 2013

ABSTRACT

The human race is growing fast. The world population is projected to top 10 billion by 2050. One response to the food supply needs of an increasing world population is creating better crop yield through crop survival. Pathogens and pests can cause catastrophic crop loss. Currently there is no widely used synthetic biological mechanism to help plants fight off bacterial pathogens and pests, so the main way large-scale growers protect crops is by using pesticides. We have engineered two possible solutions to address this problem.

In our first project, we have incorporated catalytically dead CAS9 (dCAS9) in conjunction with synthetic gRNAs complementary to pathogenic bacterial genes into a plasmid that naturally conjugates into neighboring bacterial cells. The specificity of the gRNAs will potentially allow us to target specific populations of bacterial pathogens in soil while leaving positive bacterial populations unharmed.

Our second project will provide plants with a sugar-mediated synthetic circuit that boosts a plant’s natural immune system and pesticide-producing capabilities in response to different levels of sugar a plant naturally produces in response to pests. The repression capabilities of dCAS9 will be used to regulate the synthetic circuit promoters that sense high or low sugar levels, reducing metabolic strain.

Special Thanks to our 2013 iGEM Team Sponsors!


Retrieved from "http://2013.igem.org/Team:UCSF/lily"