Team:UT-Dallas/temp

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<section id="titlechart"></html>{{Header_menu}}<html><div class="page_content"><br><h2>PROJECT</H2><p>
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<section id="titlechart"></html>{{Header_menu}}<html><div class="page_content"><br><h2>Introduction</H2><p>
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Treating infectious diseases of the gastrointestinal (GI) tract with antibiotics disrupts a patient's gut microbiota and can increase the prevalence of antibiotic resistant strains. The increasing population of multi-drug resistant bacterial strains, both within and outside of health centers, is a growing health concern that is becoming progressively difficult to treat. Additionally, it is a well-recognized fact within the global health community that traditional antibiotics do not represent a sustainable method of treatment for bacterial infections. There is a clear drive towards minimally invasive, prophylactic therapies for such ailments, but is a demand that so far, has not been adequately met. Our project will aim at replacing broad and narrow spectrum antibiotics with “precision therapies” that have etiology targeting capacity at the species level as well as contain minimal cross-talk among healthy tissues, organs, and symbiotic organisms.
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We envision a new paradigm for treating infections of the human gastrointestinal tract through exploitation of engineered probiotics that produce anti-microbials with high specificity for pathogens. The anti-microbials we are exploring do not utilize a one-fit all therapy mold, but target unique features specific to organisms at the genetic level. Towards this aim, we have utilized a general-purpose system that will be delivered to pathogenic bacteria from an engineered bacterial species found in the GI tract (Escherichia coli), which will cleave pathogenic genes with single nucleotide resolution. To achieve specific genome targeting, we will utilize the CRISPR/Cas9 system with gRNA engineered to recognize genes from infectious bacteria that contribute to pathogenicity in humans. Our CRISPR/Cas9 system will be delivered from the engineered E. coli to infectious bacteria using bacterial specific phages, minimizing any side-effects to native microbiota and human-host cells. As a proof-of-principle for our engineered probiotic, we are starting by targeting Vibrio cholerae, however we hope to expand the system to other pathogens of the GI tract.  
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<br><h2>THE TEAM</H2><br><p>
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We used E. coli as our chassis for our experiments in the lab, but we envision that our system would be used in a common probiotic strain of bacteria, such as Lactobacillus acidophilus. Our probiotic system can be used prophylactically to persons in a region experiencing a cholera outbreak, or it can be administered to someone already exposed to V. cholerae to quickly deliver therapeutic phage directly to the site of the infection. We believe our system would be particularly useful to military personnel or aid workers stationed in a cholera outbreak zone where they would be frequently exposed to V. cholerae. Although an oral cholera vaccine exists, its efficacy is relatively low: 52-62% in healthy adults and as low as 38% for the highest risk age group - children under 5 years of age (1).
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Once complete, our system will be able to remain as a stable population in a person’s gut until the event of a V. cholerae infection. Detection of V. cholerae will then activate production of the phage delivery system, which will package the gRNA and Cas9 targeting system into a phage coat, exit the probiotic and transfer the system into V. cholerae present in the gut (transmission of a heterologous DNA message via phage was demonstrated by Ortiz and Endy in 2012 and featured in Waterloo’s 2013 iGEM project (2)). Once inside V. cholerae, the targeting system will bind and cleave sites complementary to the gRNA that correspond to selected pathogenicity genes. We proposed using Cas9/gRNA to target and kill pathogens as opposed to traditional phage therapy using a targeted lytic phage because it allowed us to differentiate and kill harmful pathogens of a strain that has both harmful and harmless serotypes. The gRNA in our system, while specific to V. cholerae, can easily be altered with PCR to target unique regions in other gastrointestinal pathogens.
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  <img src="https://static.igem.org/mediawiki/2014/3/34/Sam-profile-image.png" height="400px" width="400px" class="tooltip" title="<h2 class='info_h2'>Samantha</h2>Hello! My name is Samantha and I recently graduated from UTD with a BSc in Biology. I am interested in synthetic biology because it offers very unique and dynamic solutions to complicated problems. I joined iGEM because it's an excellent platform for creative collaboration among young scientists. When I am not in the lab... it's probably because I just left to get more coffee and am on my way back to the lab.">
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  <img src="https://static.igem.org/mediawiki/2014/5/56/Megan-profile-image.png" height="400px" width="400px" class="tooltip" title="<h2 class='info_h2'>Megan Zerez</h2>Megan Zerez is a molecular biology sophomore (or junior, she’s not entirely sure) at UT Dallas. She enjoys getting used books in the mail, building things and being a food snob. She has a pet ukulele and a tendency to not attend lecture. She does not do voice modulation and likes to talk about all of these things maybe a bit too loudly, much to the chagrin of our neighbors at NSERL. In addition to wet-lab work, Megan worked on the wiki and human practices project.">
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  <img src="https://static.igem.org/mediawiki/2014/8/8b/Tra-profile-image.png" height="400px" width="400px" class="tooltip" title="<h2 class='info_h2'>Tra</h2>Tra is biology major but she is still deciding between Molecular Cell Biology and Mathematics. Nevertheless, she loves both. Since she is good at math, she does the biological system modelling for our project, despite having any prior programming experience. She learned gro and MatLab just for iGEM. Aside from wet lab work, she also helps with building the wiki. Tra loves languages. She is learning Japanese, Russian, http, and css. When there is dead time in the lab you will find her occasionally engaging in conversations on the topic of Math as a language with our labs post-doc. In between reactions, if she is not utilizing time by cleaning the lab bench, Tra would do calligraphy on the white board. Tra is an iGEM late-comer but she soon became an essential part of the team.  
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<br><br>The song ‘Tra's Brief Life as a Spider’ on Spotify reveals her life prior to iGEM. Tra was briefly reincarnated as a spider, then later, as a Wild Horse on the planet of nearly, where wild horses hold the majority in the senate. She likes pudding, but only without raisins.  
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<br><br>When not doing iGEM wet lab and dry lab work, Tra enjoys swing dancing, sacrificing bishops (in chess club), setting ants on fire with isopropanol, and slashing people with sabre (in fencing club). Tra occasionally bring borscht, mooncakes, and cashew butter to the lab.
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<br><br>Tra is at the same time engaging in a math research. She is looking to further the application of math in the study of biology.">
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  <img src="https://static.igem.org/mediawiki/2014/8/80/Rishika-profile-image.png" height="400px" width="400px" class="tooltip" title="<h2 class='info_h2'>Rishika</h2>I’m a sophomore majoring in Molecular Biology. I’m an avid Netflixer and love ice cream, napping, windy days, Chipotle, and going to airports (in that order). In lab I can most likely be found in front of the centrifuges at the Miniprep station or in front of the whiteboard admiring the lovely tables and diagrams. iGEM has been a great opportunity where I learned tons about everything ranging from the CRISPR-Cas9 system to ‘crunchy bread,’ and had a lot of fun while doing so.">
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  <img src="https://static.igem.org/mediawiki/2014/e/e7/Kortne-profile-image.png" height="400px" width="400px" class="tooltip" title="<h2 class='info_h2'>Kortne</h2>Hello, my name is Kortne Banks and I am a senior studying biology at The University of Texas at Dallas. When I am not busy studying for exams I love to spend my free time tutoring children and writing. I am currently writing a children’s book that aims towards inspiring children to pursue their dreams. A goal of mine is to one day start a nonprofit organization that helps children excel in stem careers. What prompted me to participate in Igem was my amazement on how much power the students have over choosing a project for the jamboree competition. I initially had zero bacterial cloning experience and now I am a cloning machine. I also learned a few web design techniques using html for our team wiki page. Igem has been an amazing opportunity for me and there is no doubt that this summer has been an unforgettable experience.  ">
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<p>Content 2</p>
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<br><h2>References</H2><br><p>
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1. Sinclair D., Abba K., Zaman K., Qadri F., Graves P.M., Oral vaccines for preventing cholera. Cochrane Database Syst Rev. 2011 Mar 16;(3):CD008603.<br>
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2. Ortiz M.E., Endy D. Engineered cell-cell communication via DNA messaging. J Biol Eng. 2012 Sep 7;6(1):16.</p><br><br>
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Revision as of 00:04, 18 October 2014


Introduction

We envision a new paradigm for treating infections of the human gastrointestinal tract through exploitation of engineered probiotics that produce anti-microbials with high specificity for pathogens. The anti-microbials we are exploring do not utilize a one-fit all therapy mold, but target unique features specific to organisms at the genetic level. Towards this aim, we have utilized a general-purpose system that will be delivered to pathogenic bacteria from an engineered bacterial species found in the GI tract (Escherichia coli), which will cleave pathogenic genes with single nucleotide resolution. To achieve specific genome targeting, we will utilize the CRISPR/Cas9 system with gRNA engineered to recognize genes from infectious bacteria that contribute to pathogenicity in humans. Our CRISPR/Cas9 system will be delivered from the engineered E. coli to infectious bacteria using bacterial specific phages, minimizing any side-effects to native microbiota and human-host cells. As a proof-of-principle for our engineered probiotic, we are starting by targeting Vibrio cholerae, however we hope to expand the system to other pathogens of the GI tract.




Probiotics


We used E. coli as our chassis for our experiments in the lab, but we envision that our system would be used in a common probiotic strain of bacteria, such as Lactobacillus acidophilus. Our probiotic system can be used prophylactically to persons in a region experiencing a cholera outbreak, or it can be administered to someone already exposed to V. cholerae to quickly deliver therapeutic phage directly to the site of the infection. We believe our system would be particularly useful to military personnel or aid workers stationed in a cholera outbreak zone where they would be frequently exposed to V. cholerae. Although an oral cholera vaccine exists, its efficacy is relatively low: 52-62% in healthy adults and as low as 38% for the highest risk age group - children under 5 years of age (1).

Once complete, our system will be able to remain as a stable population in a person’s gut until the event of a V. cholerae infection. Detection of V. cholerae will then activate production of the phage delivery system, which will package the gRNA and Cas9 targeting system into a phage coat, exit the probiotic and transfer the system into V. cholerae present in the gut (transmission of a heterologous DNA message via phage was demonstrated by Ortiz and Endy in 2012 and featured in Waterloo’s 2013 iGEM project (2)). Once inside V. cholerae, the targeting system will bind and cleave sites complementary to the gRNA that correspond to selected pathogenicity genes. We proposed using Cas9/gRNA to target and kill pathogens as opposed to traditional phage therapy using a targeted lytic phage because it allowed us to differentiate and kill harmful pathogens of a strain that has both harmful and harmless serotypes. The gRNA in our system, while specific to V. cholerae, can easily be altered with PCR to target unique regions in other gastrointestinal pathogens.




References


1. Sinclair D., Abba K., Zaman K., Qadri F., Graves P.M., Oral vaccines for preventing cholera. Cochrane Database Syst Rev. 2011 Mar 16;(3):CD008603.
2. Ortiz M.E., Endy D. Engineered cell-cell communication via DNA messaging. J Biol Eng. 2012 Sep 7;6(1):16.



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