Difference between revisions of "Team:Denver Biolabs/Description"

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<h3> Project Description </h3>
 
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In 2015, over 300,000 women died during pregnancy and childbirth; 99% of these deaths occurred in developing countries. Postpartum hemorrhage (PPH), or severe bleeding after birth, is the leading cause of maternal mortality worldwide. Oxytocin is a naturally occurring hormone and a medication that is used to increase contractions in the uterus (i.e., induce labor). It has also proved effective in significantly reducing the risk of PPH. Oxytocin is now routinely used in industrialized countries, and is often given in small doses simply as a preventative measure in normal labors. However, oxytocin is not readily available in developing countries. Despite being on the World Health Organization’s List of Essential Medicines for developing countries and being relatively inexpensive (as of 2014, the wholesale cost of the medication is US$0.1–0.56 per dose), oxytocin requires storage at between 2 and 8 °C, which has led to a shortage of this critical drug in rural areas that lack reliable refrigeration, power, and infrastructure. <b>Quality issues with existing oxytocin inventories have also been identified as a significant issue in rural areas.</b></p>
 
  
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<p class="main"> Our first goal is to build an oxytocin detection system using a G-protein coupled receptor in yeast that will allow us to verify the presence of active oxytocin. If successful, we will then focus our efforts on synthesizing oxytocin in various forms to increase its availability in resource-poor areas.</p>
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<p class = "main"> Oxytocin is produced in the body by the OXT gene. It is synthesized as an inactive precursor peptide along with its carrier molecule neurophysin I. After several iterations of hydrolyzation via enzymes, the active oxytocin molecule is produced. In 2013 an iGEM team from Lethbridge Canada created a form of oxytocin still attached to its carrier molecule, neurophysin I, which prevents degradation until the carrier molecule is cleaved resulting in the activation of the oxytocin molecule. Our team plans to significantly build on this prior work by exploring several other approaches to improving the stability of oxytocin including producing a powdered form of the drug that can be activated using hydrolysis, adding trace metals to prevent oxidation, and using optogenetic techniques to activate oxytocin using light. In addition to these synthetic biology approaches, the diversity of our team’s skills will allow us to explore several mechanical and hardware solutions including simple, non-refrigerated single-injection systems; biological packaging solutions to prevent oxytocin’s degradation; and <b>testing systems to evaluate the quality and effectiveness of current oxytocin inventories.</b> The solutions we plan to explore will target practices ranging from manufacturing, transportation and storage, and distribution to drug administration protocols, drug quality monitoring and control, and improved documentation and inventory to support further research and quality care.The potential impact of any and all of these solutions will be to increase the availability of oxytocin for use in under-resourced maternity facilities. </p>
 
  
  
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<figcaption style = "margin-left: 70px;padding: 10px 10px 10px;">Fig.1 - Jonah and his creepy smile</figcaption>
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  <p class ="main"> <b> Our project has three main areas of focus: <br>
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      <p class ="doubletab">Our interdisciplinary backgrounds and our convenient home within Inworks at the University of Colorado
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      Denver, allowed our team to build many of the tools we use in the lab including a fluorimeter, an optical density sensor,
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      a DNA concentration meter, an incubator, a refrigerated centrifuge, and a yeast dryer,
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      which we will be exhibiting at the Jamboree expo as part of our "Hardware" track deliverables.<br><br>
  
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<p class = "main">Successful microscopy  has confirmed the viability of EutS and EutC-eGFP in E.Coli, but the laser used to excite eGFP may also cause conformational change of azo-benzene. Instead Neptune, another light activated protein that is excited at a much longer wavelength, will be used to visualize the formation and destruction of EutS microcompartments. Future work will focus on the implementation of a multiconstruct system with EutS, EuC-Neptune, and a third construct that creates tRNA’s to integrate azo-benzene into the EutS at locations we expect to see significant steric hinderance. Instead Neptune, another light activated protein that is excited at a much longer wavelength, will be used to visualize the formation and destruction of EutS microcompartments. </p>
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<p class = "main">Future work will focus on the implementation of a multiconstruct system with EutS, EuC-Neptune, and a third construct that creates tRNA’s to integrate azo-benzene into the EutS at locations we expect to see significant steric hinderance.Instead Neptune, another light activated protein that is excited at a much longer wavelength, will be used to visualize the formation and destruction of EutS microcompartments. Future work will focus on the implementation of a multiconstruct system with EutS, EuC-Neptune, and a third construct that creates tRNA’s to integrate azo-benzene into the EutS at locations we expect to see significant steric hinderance. Future work will focus on the implementation of a multiconstruct system with EutS, EuC-Neptune, and a third construct that creates tRNA’s to integrate azo-benzene into the EutS at locations we expect to see significant steric hinderance.Future work will focus on the implementation of a multiconstruct system with EutS.  Future work will focus on the implementation of a multiconstruct system with EutS, EuC-Neptune, and a third construct that creates tRNA’s to integrate azo-benzene into the EutS at locations we expect to see significant steric hinderance. Successful microscopy  has confirmed the viability of EutS and EutC-eGFP in E.Coli, but the laser used to excite eGFP may also cause conformational change of azo-benzene. Instead Neptune, another light activated protein that is excited at a much longer wavelength, will be used to visualize the formation and destruction of EutS microcompartments. Future work will focus on the implementation of a multiconstruct system with EutS, EuC-Neptune, and a third construct that creates tRNA’s to integrate azo-benzene into the EutS at locations we expect to see significant steric hinderance. </p>
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<p class = "main">Successful microscopy has confirmed the viability of EutS and EutC-eGFP in E.Coli, but the laser used to excite eGFP may also cause conformational change of azo-benzene. Instead Neptune, another light activated protein that is excited at a much longer wavelength, will be used to visualize the formation and destruction of EutS microcompartments. Future work will focus on the implementation of a multiconstruct system with EutS, EuC-Neptune, and a third construct that creates tRNA’s to integrate azo-benzene into the EutS at locations we expect to see significant steric hinderance. Instead Neptune, another light activated protein that is excited at a much longer wavelength, will be used to visualize the formation and destruction of EutS microcompartments. Future work will focus on the implementation of a multiconstruct system with EutS, EuC-Neptune, and a third construct that creates tRNA’s to integrate azo-benzene into the EutS at locations we expect to see significant steric hinderance.Instead Neptune, another light activated protein that is excited at a much longer wavelength, will be used to visualize the formation and destruction of EutS microcompartments. Future work will focus on the implementation of a multiconstruct system with EutS, EuC-Neptune, and a third construct that creates tRNA’s to integrate azo-benzene into the EutS at locations we expect to see significant steric hinderance. Future work will focus on the implementation of a multiconstruct system with EutS, EuC-Neptune, and a third construct that creates tRNA’s to integrate azo-benzene into the EutS at locations we expect to see significant steric hinderance. Future work will focus on the implementation of a multiconstruct system with EutS, EuC-Neptune, and a third construct that creates tRNA’s to integrate azo-benzene into the EutS at locations we expect to see significant steric hinderance.</p>
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<p class = "main"> Successful microscopy  has confirmed the viability of EutS and EutC-eGFP in E.Coli, but the laser used to excite eGFP may also cause conformational change of azo-benzene. Instead Neptune, another light activated protein that is excited at a much longer wavelength, will be used to visualize the formation and destruction of EutS microcompartments. Future work will focus on the implementation of a multiconstruct system with EutS, EuC-Neptune, and a third construct that creates tRNA’s to integrate azo-benzene into the EutS at locations we expect to see significant steric hinderance. </p>
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      The fluorimeter and optical density sensor we built was based on a design by the <A HREF = "https://2014.igem.org/Team:Aachen/OD/F_device">2014 Aachen iGEM team</A>.
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      We improved upon the original design by designing a 3-in-1 cuvette holder using a Form 2 Resin 3D printer.
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<h5>Before you start: </h5>
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      Different colored filters can be inserted interchangeably into the cuvette holder to filter
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<p> Please read the following pages:</p>
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      different frequencies of light, and thus detect opitcal density or fluoresence or DNA concentration.
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<ul>
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      We also improved upon the original Arduino code by refactoring and commenting functions. We also used
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<li>  <a href="https://2016.igem.org/Requirements">Requirements page </a> </li>
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      a four-button Olimex LCD screen, which required re-mapping functions to the four different buttons, and
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<li> <a href="https://2016.igem.org/Wiki_How-To">Wiki Requirements page</a></li>
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      adding DNA concentration calculations to the code base. All of our code is open source and available for download
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<li> <a href="https://2016.igem.org/Resources/Template_Documentation"> Template Documentation </a></li>
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      on our <a href="https://github.com/denverbiolabs/">github repository.</a><br><br>
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      The high-level goal of our project is make biological diagnostics and tools more accessible in
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      resource-constrained areas. The difficulty with implementing biological diagnostic tools in these places, however,
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      is largely the same as the issues that orginally motivated our project: lack of refrigeration and resources.
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<p>You may style this page as you like or you can simply leave the style as it is. You can easily keep the styling and edit the content of these default wiki pages with your project information and completely fulfill the requirement to document your project.</p>
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      We therefore wanted to design a system where engineered yeast could be "dried" similar to the process for
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<p>While you may not win Best Wiki with this styling, your team is still eligible for all other awards. This default wiki meets the requirements, it improves navigability and ease of use for visitors, and you should not feel it is necessary to style beyond what has been provided.</p>
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      bakers and brewers yeast, shipped in inexpensive packets at room temperature, and reconstituted on site
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      to preform the intended diagnostic funtion. To meet this goal we built a yeast drier, modeled after
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      industrial yeast driers. Our <a href="https://2016.igem.org/Team:Denver_Biolabs/Design">Project Design</a> and
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      <a href="https://2016.igem.org/Team:Denver_Biolabs/Proof">Proof of Concept</a> pages contain more details
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      on the construction and implementation of our hardware designs.
  
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      <!-- INSERT PHOTO OF YEAST DRIER-->
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<h5> Wiki template information </h5>
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<p>We have created these wiki template pages to help you get started and to help you think about how your team will be evaluated. You can find a list of all the pages tied to awards here at the <a href="https://2016.igem.org/Judging/Pages_for_Awards/Instructions">Pages for awards</a> link. You must edit these pages to be evaluated for medals and awards, but ultimately the design, layout, style and all other elements of your team wiki is up to you!</p>
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      <p class ="tab"> <b>2) Create an oxytocin quality sensor using a G-protein coupled receptor in yeast:</b></p>
  
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      <p class ="doubletab"> The second goal of our project was to design and test a diagnostic system for medication quality
 +
        that leveraged G-protein coupled receptors (GPCRs) in yeast. We relied on the DIY receptor design instructions created by
 +
        <a href="https://2012.igem.org/Team:TU-Delft/receptordesign">TU-Delft's 2012 iGEM team</a> to create our own
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        oxytocin GPCR. TU-Delft's design leveraged work done by <i>Radhika et. al.</i>
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        who engineered <i>Saccharomyces cerevisiae</i> to contain components of the mammalian (rat) olfactory signaling
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        pathway to detect dinitrotoluene, an explosive residue. TU-Delft used this design to create
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        <i>Snifferomyces</i>, a yeast strain capable of detecting Niacin, a compound exhaled by individuals
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        with tuberculosis. Our goal was to use the same GPCR approach to design yeast that could detect
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        active oxytocin in order to determine if the medication sample was expired or not. <br><br>
  
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        We began by designing our receptor using SnapGene (one of our generous sponsors) and having it
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        synthesized by IDT (for free as part of their sponsorship of iGEM 2016, thank you!) We then cloned
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        our OXTR part into the TU-Delft part from the BioBrick distribution kit to create a composite part.
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        We used 3A assembly to remove the composite part which included a yeast promoter, a RBS, our OXTR part,
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        a terminator, and the FUS-1 and EGFP parts from TU-Delft.<br><br>
  
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        Our next step was to order several shuttle vectors from AddGene so we could produce more of our part
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<h5> Editing your wiki </h5>
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        using E.coli, mini-prep it out, and transform it into yeast where it would express the proteins required for
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<p>On this page you can document your project, introduce your team members, document your progress and share your iGEM experience with the rest of the world! </p>
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        our oxytocin receptor to form on the cell's surface, and when oxytocin is detected, trigger the FUS-1 mating
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<p> <a href="https://2016.igem.org/wiki/index.php?title=Team:Example&amp;action=edit"> </a>Use WikiTools - Edit in the black menu bar to edit this page</p>
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        pathway and produce measureable green fluorescent protein.<br><br>
  
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</div>
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        In our work on this aspect of the project we experimented with different reporters, yeast promoters and temrinators,
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        yeast strains, and selection techniques. More details can be found on our <a href="https://2016.igem.org/Team:Denver_Biolabs/Design">Project Design</a> and
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        <a href="https://2016.igem.org/Team:Denver_Biolabs/Proof">Proof of Concept</a> pages.
  
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      </p>
  
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      <p class ="tab"> <b>3) Produce a more temperature stable form of oxytocin using E.coli and/or yeast:</b></p>
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<h5>Tips</h5>
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<p>This wiki will be your team’s first interaction with the rest of the world, so here are a few tips to help you get started: </p>
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<ul>
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<li>State your accomplishments! Tell people what you have achieved from the start. </li>
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<li>Be clear about what you are doing and how you plan to do this.</li>
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<li>You have a global audience! Consider the different backgrounds that your users come from.</li>
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<li>Make sure information is easy to find; nothing should be more than 3 clicks away. </li>
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<li>Avoid using very small fonts and low contrast colors; information should be easy to read.  </li>
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<li>Start documenting your project as early as possible; don’t leave anything to the last minute before the Wiki Freeze. For a complete list of deadlines visit the <a href="https://2016.igem.org/Calendar">iGEM 2016 calendar.</a> </li>
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<li>Have lots of fun! </li>
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</ul>
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      <p class ="doubletab"> The third, and original goal, of our project was to directly address the
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      short half-life and need for refrigeration of the life-saving drug oxytocin. We have begun work on this
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      aspect of the project in several ways. First, we researched E.coli strains capable of folding human proteins,
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      and ordered BL21. We also began testing different compentent cell protocols to maximize our transformation
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      efficiency of this strain. We also requested a part iGEM HQ
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      submitted by the <a href="https://2013hs.igem.org/Team:Lethbridge_Canada/project"> Lethbridge Canada 2013 iGEM team </a>.
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      The Lethbridge part included the oxytocin gene and its precursor molecule nueorphysin-I. The goal of the Lethbridge
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      team was also to create a longer-lasting form of oxytocin by adding precursor molecules to slow the hormone's degradation.
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      We are working to improve upon their part by experimenting with different secretion tags to facilitate easier
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      precipitation of the resulting protein. <br><br>
  
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      We are still working on this aspect of the project, researching possible mechanisms for producing
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<h5>Inspiration</h5>
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      the human oxytocin protein using yeast, and exploring possible temperature regulation mechanisms to
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<p> You can also view other team wikis for inspiration! Here are some examples:</p>
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      the instability of this essential medication.
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<ul>
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<li> <a href="https://2014.igem.org/Team:SDU-Denmark/"> 2014 SDU Denmark </a> </li>
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<li> <a href="https://2014.igem.org/Team:Aalto-Helsinki">2014 Aalto-Helsinki</a> </li>
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<li> <a href="https://2014.igem.org/Team:LMU-Munich">2014 LMU-Munich</a> </li>
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<li> <a href="https://2014.igem.org/Team:Michigan"> 2014 Michigan</a></li>
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<li> <a href="https://2014.igem.org/Team:ITESM-Guadalajara">2014 ITESM-Guadalajara </a></li>
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<li> <a href="https://2014.igem.org/Team:SCU-China"> 2014 SCU-China </a></li>
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</ul>
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</div>
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<h5> Uploading pictures and files </h5>
 
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<p> You can upload your pictures and files to the iGEM 2016 server. Remember to keep all your pictures and files within your team's namespace or at least include your team's name in the file name. <br />
 
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When you upload, set the "Destination Filename" to <br><code>T--YourOfficialTeamName--NameOfFile.jpg</code>. (If you don't do this, someone else might upload a different file with the same "Destination Filename", and your file would be erased!)</p>
 
  
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  <li><a href="https://2016.igem.org/Team:Denver_Biolabs/Composite_Part"> ★ Composite Parts </a></li>
 
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                    © 2016 Denver Biolabs 501(c)(3) <br>All rights reserved
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Revision as of 21:06, 9 October 2016

Team:Denver_Biolabs - 2016.igem.org

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Team:Denver_Biolabs

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Project Description


Our project has three main areas of focus:

1) Design and build low-cost open source biotools:

Our interdisciplinary backgrounds and our convenient home within Inworks at the University of Colorado Denver, allowed our team to build many of the tools we use in the lab including a fluorimeter, an optical density sensor, a DNA concentration meter, an incubator, a refrigerated centrifuge, and a yeast dryer, which we will be exhibiting at the Jamboree expo as part of our "Hardware" track deliverables.

The fluorimeter and optical density sensor we built was based on a design by the 2014 Aachen iGEM team. We improved upon the original design by designing a 3-in-1 cuvette holder using a Form 2 Resin 3D printer. Different colored filters can be inserted interchangeably into the cuvette holder to filter different frequencies of light, and thus detect opitcal density or fluoresence or DNA concentration. We also improved upon the original Arduino code by refactoring and commenting functions. We also used a four-button Olimex LCD screen, which required re-mapping functions to the four different buttons, and adding DNA concentration calculations to the code base. All of our code is open source and available for download on our github repository.

The high-level goal of our project is make biological diagnostics and tools more accessible in resource-constrained areas. The difficulty with implementing biological diagnostic tools in these places, however, is largely the same as the issues that orginally motivated our project: lack of refrigeration and resources. We therefore wanted to design a system where engineered yeast could be "dried" similar to the process for bakers and brewers yeast, shipped in inexpensive packets at room temperature, and reconstituted on site to preform the intended diagnostic funtion. To meet this goal we built a yeast drier, modeled after industrial yeast driers. Our Project Design and Proof of Concept pages contain more details on the construction and implementation of our hardware designs.

2) Create an oxytocin quality sensor using a G-protein coupled receptor in yeast:

The second goal of our project was to design and test a diagnostic system for medication quality that leveraged G-protein coupled receptors (GPCRs) in yeast. We relied on the DIY receptor design instructions created by TU-Delft's 2012 iGEM team to create our own oxytocin GPCR. TU-Delft's design leveraged work done by Radhika et. al. who engineered Saccharomyces cerevisiae to contain components of the mammalian (rat) olfactory signaling pathway to detect dinitrotoluene, an explosive residue. TU-Delft used this design to create Snifferomyces, a yeast strain capable of detecting Niacin, a compound exhaled by individuals with tuberculosis. Our goal was to use the same GPCR approach to design yeast that could detect active oxytocin in order to determine if the medication sample was expired or not.

We began by designing our receptor using SnapGene (one of our generous sponsors) and having it synthesized by IDT (for free as part of their sponsorship of iGEM 2016, thank you!) We then cloned our OXTR part into the TU-Delft part from the BioBrick distribution kit to create a composite part. We used 3A assembly to remove the composite part which included a yeast promoter, a RBS, our OXTR part, a terminator, and the FUS-1 and EGFP parts from TU-Delft.

Our next step was to order several shuttle vectors from AddGene so we could produce more of our part using E.coli, mini-prep it out, and transform it into yeast where it would express the proteins required for our oxytocin receptor to form on the cell's surface, and when oxytocin is detected, trigger the FUS-1 mating pathway and produce measureable green fluorescent protein.

In our work on this aspect of the project we experimented with different reporters, yeast promoters and temrinators, yeast strains, and selection techniques. More details can be found on our Project Design and Proof of Concept pages.

3) Produce a more temperature stable form of oxytocin using E.coli and/or yeast:

The third, and original goal, of our project was to directly address the short half-life and need for refrigeration of the life-saving drug oxytocin. We have begun work on this aspect of the project in several ways. First, we researched E.coli strains capable of folding human proteins, and ordered BL21. We also began testing different compentent cell protocols to maximize our transformation efficiency of this strain. We also requested a part iGEM HQ submitted by the Lethbridge Canada 2013 iGEM team . The Lethbridge part included the oxytocin gene and its precursor molecule nueorphysin-I. The goal of the Lethbridge team was also to create a longer-lasting form of oxytocin by adding precursor molecules to slow the hormone's degradation. We are working to improve upon their part by experimenting with different secretion tags to facilitate easier precipitation of the resulting protein.

We are still working on this aspect of the project, researching possible mechanisms for producing the human oxytocin protein using yeast, and exploring possible temperature regulation mechanisms to the instability of this essential medication.

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