Team:Stony Brook/Team

From 2014.igem.org

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</head>
</head>
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<body bgcolor="#ffffff">
 
-
<div id="yellow_header">  <a href="http://www.stonybrook.edu/">Stony Brook University</a></div>
 
 +
<body>
 +
<div id="yellow_header"> <a href="http://www.stonybrook.edu/">Stony Brook University</a></div>
<div id="blue_header"> <a href="https://2014.igem.org/Team:Stony_Brook"><img src="https://static.igem.org/mediawiki/2014/1/1e/Stony_Brook_Apis-biotics_header.png"/ id="apis-biotics"/></a>
<div id="blue_header"> <a href="https://2014.igem.org/Team:Stony_Brook"><img src="https://static.igem.org/mediawiki/2014/1/1e/Stony_Brook_Apis-biotics_header.png"/ id="apis-biotics"/></a>
-
 
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  <div id="yellow_navigation_container">
-
<div id="yellow_navigation_container">
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    <div id="yellow_navigation"> <a href="https://2014.igem.org/Team:Stony_Brook"><img src="https://static.igem.org/mediawiki/2014/7/78/Stony_Brook_NavIcon2_Home.png"/></a>
-
+
      <p> Home </p>
-
<div id="yellow_navigation"> <a href="https://2014.igem.org/Team:Stony_Brook"><img src="https://static.igem.org/mediawiki/2014/7/78/Stony_Brook_NavIcon2_Home.png"/></a>
+
    </div>
-
  <p> Home </p> </div>
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-
   
+
     <div id="yellow_navigation"> <a href="https://2014.igem.org/Team:Stony_Brook/Team"><img src="https://static.igem.org/mediawiki/2014/6/66/Stony_Brook_NavIcon2_Team.png"/></a>
     <div id="yellow_navigation"> <a href="https://2014.igem.org/Team:Stony_Brook/Team"><img src="https://static.igem.org/mediawiki/2014/6/66/Stony_Brook_NavIcon2_Team.png"/></a>
-
    <p> Team </p>
+
      <p> Team </p>
     </div>
     </div>
-
       
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    <div id="yellow_navigation"> <a href="https://2014.igem.org/Team:Stony_Brook/Project"><img src="https://static.igem.org/mediawiki/2014/9/99/Stony_Brook_NavIcon2_Project.png"/></a>
-
    <div id="yellow_navigation"> <a href="https://2014.igem.org/Team:Stony_Brook/Project"><img src="https://static.igem.org/mediawiki/2014/9/99/Stony_Brook_NavIcon2_Project.png"/></a>
+
      <p> Project </p>
-
    <p> Project </p>
+
    </div>
 +
    <div id="yellow_navigation"> <a href="https://2014.igem.org/Team:Stony_Brook/Notebook"><img src="https://static.igem.org/mediawiki/2014/5/57/Stony_Brook_NavIcon2_Notebook.png"/></a>
 +
      <p> Notebook </p>
 +
    </div>
 +
<div id="yellow_navigation">
 +
  <a href="https://2014.igem.org/Team:Stony_Brook/Results"> <img src="https://static.igem.org/mediawiki/2014/0/04/Stony_Brook_NavIcon2_Results.png"/></a>
 +
      <p>Results</p>
 +
</div>
 +
    <div id="yellow_navigation"> <a href="https://2014.igem.org/Team:Stony_Brook/Outreach"><img src="https://static.igem.org/mediawiki/2014/8/80/Stony_Brook_NavIcon2_Outreach.png"/></a>
 +
      <p> Outreach </p>
 +
    </div>
 +
<div id="yellow_navigation">
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  <a href="https://2014.igem.org/Team:Stony_Brook/Safety"> <img src="https://static.igem.org/mediawiki/2014/0/07/Stony_Brook_NavIcon2_Safety.png"/></a>
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      <p>Safety</p>
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    <div id="yellow_navigation"> <a href="https://2014.igem.org/Team:Stony_Brook/Attributions"><img src="https://static.igem.org/mediawiki/2014/0/03/Stony_Brook_NavIcon2_Acknowledgements.png"/></a>
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      <p> Attributions </p>
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  <p> Outreach </p>
 
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<p> Attributions </p>
 
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<div id="project"><p>Project</p>  
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  <p>Meet the Team!</p>
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<div id="abstract"> <h3>Abstract</h3>
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<p>Pathogenic bacteria are becoming increasingly antibiotic-resistant due to misuse, overuse and abuse. In addition, infections caused by some harmful strains of bacteria, particularly gram-negative bacteria, cannot easily be treated with antibiotics or other common forms of treatment. Our intention is to engineer cells which can both produce an antimicrobial peptide and recognize the communication signals of such bacteria, using Pseudomonas aeruginosa as a model. Two plasmids, one which controls the production of our antimicrobial peptide melittin and the other which acts as the cell-signal receiver, will be inserted in nonpathogenic E. coli cells. This allows our E. coli to recognize the cells of Pseudomonas aeruginosa, and release a bacteria-killing compound in response. </p>
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  <p> Our advisors </p>
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<div id="gergen"><figure><img src="https://static.igem.org/mediawiki/2014/f/fa/Stony_Brook_TeamAdvisors_Gergen.png"/><figcaption>John Peter Gergen<br /><i>Department of<br />Biochemistry and<br />Cell Biology</i></figcaption></figure></div>
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    <label for="tab-4" class="tab-label-4">Our Solution: Apis-Biotics</label>
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            <h4>WHEN ANTIBIOTICS FAIL</h4>
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When Alexander Fleming was accepting his Nobel Prize in medicine for his discovery of penicillin, the world’s first antibiotic, he warned of a future where antibiotics are no longer effective. <sup><a href="#1">[1]</a></sup> The growth of antibacterial resistance—when bacteria no longer are affected by antibiotics—is quickly becoming a major public health concern. In 2014, the World Health Organization stated “A post-antibiotic era—in which common infections and minor injuries can kill—far from being an apocalyptic fantasy, is instead a very real possibility for the 21<sup>st</sup> century.” <sup><a href="#2">[2]</a></sup> </p>
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<div id="czaplinski"><figure><img src="https://static.igem.org/mediawiki/2014/5/53/Stony_Brook_TeamAdvisors_Czaplinski.png"/><figcaption>Kevin Czaplinski<br /><i>Department of<br />Biochemistry and<br />Cell Biology</i></figcaption></figure></div>
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<h4>THE MECHANISM OF ANTIBIOTIC RESISTANCE</h4>
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<div id="balazsi"><figure><img src="https://static.igem.org/mediawiki/2014/b/bc/Stony_Brook_TeamAdvisors_Balazsi.png"/><figcaption>Gabor Balazsi<br /><i>Department of<br />Biomedical Engineering</i></figcaption></figcaption></figure></div>
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<p>Antibiotic resistance is a natural phenomenon that occurs when mutations in bacterial replications render antibiotics ineffective. Many antibiotics kill bacteria by either preventing
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them from creating bacterial cell walls, affecting their DNA, RNA, or proteins, or changing their metabolism. However, through mutations, bacteria may develop the ability to break down <figure id="betalactamase">
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<figcaption>Bacteria are constantly rebuilding their cell walls through peptidoglycan synthesis. Penicillin works by deactivating transpeptidase within the bacteria, preventing the cross-linking of peptidoglycan and weakening the cell wall, eventually causing lysis due to osmotic pressure. Bacteria can develop resistance to penicillin by producing β-lactamase, which breaks the beta-lactam ring in penicillin, deactivating it.</figcaption></figure>these antibiotics, prevent the antibiotic from entering the cell, or use efflux pumps in the cell membrane to transport the antibiotic back outside the cell. When antibiotics are used to kill off harmful bacteria, the resistant strain of bacteria remains and reproduces. With other strains
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<div id="chan"><figure><img src="https://static.igem.org/mediawiki/2014/f/fb/Stony_Brook_TeamAdvisors_Chan.png"/><figcaption>Ete Chan<br /><i>Department of<br />Biomedical Engineering</i></figcaption></figure></div>
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<p>However, what occurs more often than mutation-derived resistance is the spread of antibiotic resistance through horizontal gene transfer, which occurs when genetic material from bacteria, such as plasmids, transposons, or DNA are passed from cell to cell. Through this mechanism, bacteria can even develop multi-drug resistance. Even when a singular antibiotic is used for more than ten days, multidrug resistance develops to structurally unrelated drugs as the resistant bacteria recruit resistance genes from other bacteria in the environment, encouraging the development of “super-bugs.” In addition, some bacteria are intrinsically resistant to bacteria, such as gram-negative bacteria, which due to its thicker outer cell membrane are harder to treat with certain classes of antibiotics. In some cases, some strains of gram-negative bacteria are resistant to all antibiotics, even the
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<div id="meng"><figure><img src="https://static.igem.org/mediawiki/2014/6/69/Stony_Brook_TeamAdvisors_Meng.png"/><figcaption>Yizhi Meng<br /><i>Department of<br />Materials Science<br />and Engineering</i></figcaption></figure></div>
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newest families of antibiotics and antibiotics used as a last resort.  <sup><a href="#3">[3]</a></sup>
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<figure id="antibioticresistance"><img src="https://static.igem.org/mediawiki/2014/c/ce/Stony_Brook_Project_Antibiotic_resistance_selection.png"><figcaption>After an antibiotic is used, bacteria with resistance genes are selected while those without resistance die off, allowing resistant bacteria to better compete for resources and thrive</figcaption></figure><figure id="horizontal"><img src="https://static.igem.org/mediawiki/2014/4/43/Stony_Brook_Project_Horizontal_gene_transfer.png"><figcaption> Horizontal gene transfer: DNA for antibiotic resistance is transfered through conjugation.</figcaption></figure>
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  <p> Team Members </p>
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<p>The misuse of antibiotics in the healthcare environment contributes one avenue for the spread of antibiotic resistance. In one study done for the CDC, researchers found that almost 80% of hospitals misuse antibiotics, either giving inappropriate or redundant antibiotic combinations and intravenous antibiotics. <sup><a href="#4">[4]</a></sup> The use of antibiotics in food and agriculture also presents a significant problem. The Union of Concerned Scientists estimated in one report that 70% of all antibiotics used in the U.S. are used in the food and water of already-healthy livestock, in order to control disease. Many of these antibiotics don’t break down in the waste of livestock, leading to the growth of resistant bacteria which spreads as this waste is used as fertilizer, contaminating the soil and sources of water. This contamination can also be found coming from antibiotic manufacturing plants, as well as households which improperly dispose of unused or expired
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antibiotic pills, instead or returning them to a pharmacy. <sup><a href="#4">[5]</a></sup></p>
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<h4>TO WHAT EXTENT?</h4>
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<p>In its 2014 report on antibiotic resistance, the World Health Organization reported the current status of antibiotic resistance in seven major bacteria which cause disease in humans, including Escherichia coli, Klebsiella pneumonia, Staphylococcus aureus, and Streptococcus pneumoniae. These bacteria are common causes of infections within a hospital or community. If they become highly resistant to antibiotics, there would be significant repercussions for public health
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worldwide. </p>
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<img src="https://static.igem.org/mediawiki/2014/7/72/Stony_Brook_TeamHeadshot_Maryam.png" onmouseover="document.getElementById('maryam').style.display='block';"  onmouseout="document.getElementById('maryam').style.display='none';"/>
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<img src="https://static.igem.org/mediawiki/2014/3/3b/Stony_Brook_TeamHeadshot_Karen.png" onmouseover="document.getElementById('karen').style.display='block';"  onmouseout="document.getElementById('karen').style.display='none';"/>
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<img src="https://static.igem.org/mediawiki/2014/e/e7/Stony_Brook_TeamHeadshot_Kamal.png" onmouseover="document.getElementById('kamal').style.display='block';"  onmouseout="document.getElementById('kamal').style.display='none';"/>
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<figure class="center"><img src='https://static.igem.org/mediawiki/2014/b/bc/Stony_Brook_Project_Antibiotic_resistance_chart.png'><figcaption> Data taken from WHO 2014 report on antimicrobial resistance </figcaption></figure>
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<h4>WHY DOES IT MATTER?</h4>
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<p>Antibiotic resistance is a global concern—it jeopardizes our ability to treat common infections, causing standard treatments to be replaced by more expensive drug therapies, making infectious diseases longer to treat and easier to spread, putting major surgeries, chemotherapy and organ transplants at a greater risk of being unsuccessful, and increasing death rate. <sup><a href="#2">[2]</a></sup> MRSA, or methicillin-resistant Staphycoccus aureus, and drug-resistant Staphococcus aureus have already
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been categorized as “serious threats” by the CDC. <sup><a href="#6">[6]</a></sup>The increasing resistance of these bacteria to the latest generation of antibiotics eventually requires broader spectrum- drugs which have higher costs, higher risks and worse side effects to patients due to a more toxic treatment. The growth of antibiotic resistance in pathogenic bacteria renders one of our most powerful tools against disease useless—making it imperative to develop new courses of treatment.</p>
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<img src="https://static.igem.org/mediawiki/2014/4/44/Stony_Brook_TeamHeadshot_Millie.png" onmouseover="document.getElementById('millie').style.display='block';"  onmouseout="document.getElementById('millie').style.display='none';"/>
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<img src="https://static.igem.org/mediawiki/2014/4/4a/Stony_Brook_TeamHeadshot_Tenzing.png" onmouseover="document.getElementById('tenzing').style.display='block';"  onmouseout="document.getElementById('tenzing').style.display='none';"/>
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<p><h4>REFERENCES</h4>
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<div class="photo"><img src="https://static.igem.org/mediawiki/2014/7/7e/Stony_Brook_TeamFullshot_Greg.png"/></div>
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<a id="1"></a> <li> Fleming, Alexander. "Nobel Lecture: Penicillin." Speech, Nobel Prize Award Ceremony from Nobel Foundation, Stockholm, December 11, 1945.</li>
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    <div class="bio"><h1>Gregory Poterewicz</h1><p>Year: Junior<br>Major: Biology</p><p>When I finally get to leave the lab, I go to sleep for the few hours that I have. When I actually have time, I love to go out into the world and explore, always looking to have new experiences. I am hoping to go to grad school after Stony Brook where my research will conveniently take me to exotic places.</p><p><i>Favorite Lab Moment:</i><br />When we found out about our anonymous donor because it meant that the work we were putting in meant something to someone.</p></div>
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<a id="2"></a><li> World Health Organization. Antimicrobial Resistance: Global Report on Surveillance 2014. S.l.:  
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    <div class="bio"><h1>Helen Liu</h1><p>Year: Sophomore<br>Major: Chemical Engineering</p><p>I've often been told I have a surprising sense of humor. Sometimes I even surprise myself. In my free time, I like to watch TV, or sleep, or fall asleep while watching TV. After helping to build the wiki, I'm looking forward to not looking at HTML/CSS for very long time.<p><i>Favorite Lab Moment:</i><br />When Nic, Gurkamal and I masterminded a kidnapping of Gouda's origami cranes when he went on vacation. (But we all know the real mastermind was me.)</p></div>
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World Health Organization, 2014.</li>
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    <div class="bio"><h1>Janki Patel</h1><p>Year: Sophomore<br>Major: Biochemistry</p><p>One day I plan to one day attend medical school and eventually achieve a career as a doctor.  After shadowing around the medical field for a year, the study of the brain has particularly fascinated me, so neurology is a field that I'm looking to pursue.  While  attending Stony Brook, I will further my knowledge in these areas and contribute to my community through both volunteering and research.<p><i>Favorite Lab Moment:</i><br />The first time I ever touched anything in the lab. A gel crumbled in my hands and I was afraid to do any lab work for a solid month</p></div>
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<a id="3"></a><li> Levy, Stuart B, and Bonnie Marshall. "Antibacterial Resistance Worldwide: Causes, Challenges And
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    <div class="bio"><h1>Maryam Ige</h1><p>Year: Junior<br>Major: Biomedical Engineering</p><p>I’ve often been told that I am a challenge driven person. I can’t help that I relish the feeling of accomplishment. So I’m often in situations where I am testing my limits. But what THE ultimate challenge to me is, is dabbling in the impossible; having my imagination brought to life. Hence, iGEM was like a blessing for me: it empowered with the opportunity to create biological systems not found in nature. So cool!!!!</p><p><i>Favorite Lab Moment:</i><br />When I became a pro in origami. Thanks Helen!</p></div>
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Responses." Nature Medicine 10, no. 12s (2004): S122-S129.</li>
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    <div class="bio"><h1>Gurkamal Kaur</h1><p>Year: Junior<br>Major: Biology</p><p>Hello my name is Gurkamal Kaur! Here at Stony Brook, I am pursuing a career in medicine and hope to become a future educator in the medical field. Every summer, I participate in a summer day camp and teach elementary school children reading and math skills. It is quite satisfying to know you are making a difference in a child's life and inspiring them to learn while having fun!  In my free time, I like to engage in long distance running and playing the lovely sport of volleyball! Above all, there is nothing quite like going hiking with my amazing dog, Oreo! </p><p><i>Favorite Lab Moment:</i><br />Streaming the FIFA world cup in lab. Talk about multi-tasking! </p></div>
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<a id="4"></a><li> Schultz L, Lowe TJ, Srinivasan A, Neilson D, Pugliese G. “Economic Impact of Redundant
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    <div class="bio"><h1>Karen Wong</h1><p>Year: Junior<br>Major: Biology<br>Minor: Biomedical Engineering</p><p>I'm on the Pre-Med track, but after joining the iGEM team, I realized that I should keep an open mind to graduate school or a career in different areas of science and medicine. I am very outspoken about my thoughts on feminism and the equality between men and women, but I am constantly learning new perspectives and ideas that add to my knowledge of the issue. I want boys and girls to grow up with as few differences as possible.</p><p><i>Favorite Lab Moment:</i><br />Debating with Greg as to why I don’t like the term “chick flick.”</div>
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Antimicrobial Therapy in US Hospitals.” Infection Control and Hospital Epidemiliogy 35: 1229-1235.</li>
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    <div class="bio"><h1>Mohamed Gouda</h1><p>Year: Junior<br>Major: Biochemistry</p><p>I hold a number of interests outside of studying and pursuing a career in medicine. High on that list would have to be chess. I enjoy playing, learning, and even watching games of chess. I'm also a fan of watching films; drama, comedy, thriller, you name it, I enjoy it. </p><p><i>Favorite Lab Moment:</i><br />That one time I did something so cool Nic ripped his shorts.</p></div>
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<a id="5"></a><li>. Rosenblatt-Farrell N. “The Landscape of Antibiotic Resistance.” Environ Health Perspect. 117(6):  
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    <div class="bio"><h1>Andrew Xu</h1><p>Year: Sophomore<br>Major: Biology</p><p>My absolute favorite thing to do in the world is to take a nap. I never quite get enough sleep as a pre-medical student. Of course, I still love studying for my science courses because I aspire to practice in the field of sports medicine. In between the long arduous hours at the library, I do enjoy training as a powerlifter in the gym and hunting for my next junk food fix. But, if you see me, I will love you very much if you can spare a cup of black coffee or an energy drink!</p><p><i>Favorite Lab Moment:</i><br />That one day Helen didn't hate me.</p></div>
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A244-A250.</li>
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    <div class="bio"><h1>Nicolai Tayco</h1><p>Year: Sophomore<br>Major: Biochemistry<br>Minor: Studio Art</p><p>My dream, aside from continuing research and going to medical school, is to is to pursue my love of art. I initially planned on droppping it after high school so I can focus on my major, but I just couldn't stay away from it. I've heard a lot of arguments for the discontinuation of art education in schools, but the arts and sciences go hand-in-hand more than people realize. I believe in a well-rounded education for future generations.</p><p><i>Favorite Lab Moment:</i><br />That one time Gouda did something so cool I ripped my shorts.</p></div>
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<a id="6"></a><li> Centers for Disease Control and Prevention. Antibiotic resistance threats in the United States, 2013.  
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S.l.: Centers for Disease Control and Prevention, 2013.</li></ol></p>
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    <div class="bio"><h1>Tenzing Lama</h1><p>Year: Senior<br>Major: Biochemistry<br>Minor: Chemistry</p><p>Hi! My name is Tenging Lama. After taking several upper level science courses, and working in an organic synthesis laboratory, I have developed a serious knack of learning more sciences, and I am extremely interested in pursuing my own independent research sometime in the future. In my free time, I like to get together with my good friends and travel to new exciting places. </p><p><i>Favorite Lab Moment:</i><br />“That’s it!” – Only few would understand this catchphrase!</p></div>
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<h4>OUR MODEL: PSEUDOMONAS AERUGINOSA</h4>
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<p>Pseudomonas aeruginosa is an example of an opportunistic and virulent pathogen which is strongly resistant against antibiotics. In addition to its developed antibiotic resistance, P. aeruginosa has intrinsic resistance to antibiotics, due to its low membrane permeability as well as pumps in its membrane which actively pump out antibiotics from the cell.<sup><a href="#8">[1]</a></sup> Multi-drug resistant P. aeruginosa is particularly prevalent in hospitals, where it can colonize in hospital equipment and cause cross-infections in patients with weakened immune systems, such as in patients with cystic fibrosis. <sup><a href="#9">[2]</a></sup>  
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<figure id="pa"> <img src="https://static.igem.org/mediawiki/2014/d/dd/Stony_Brook_Project_Paeruginosa.jpg" height="300" width="300"><figcaption> Biofilm formation of P. aeruginosa (<a href="http://www.uoguelph.ca/~confocal/gallery/Image027Snapshot1.jpg"> Source </a>).</figcaption></figure>
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Serious P. aeruginosa infections can cause meningitis, pneumonia, and sepsis. <sup><a href="#10">[3]</a></sup> The CDC estimates that 51,000 healthcare-associated infections in the US are caused by P. aeruginosa, 13% of which are multidrug resistant and which the CDC considers a serious threat. <sup><a href="#11">[4]</a></sup> In addition,  30% of  healthcare-related P. aeruginosa infections are resistant to fluoroquinolones, a broad-spectrum antibiotic. <sup><a href="#12">[5]</a></sup> The resistance of P. aeruginosa can therefore become deadly for immunocompromised patients, leading to higher mortality rates for affected patients.</p>
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    <div class="bio"><h1>Millicent Mulieri</h1><p>Year: Sophomore<br>Major: Biochemistry<br>Minor: Political Science</p><p>My name is Millicent Mulieri, but I go by Millie. I am an active member of the Global Medical Brigades and plan on joining Doctors Without Borders upon completion of medical school. My ultimate goal is to set up free healthcare clinics throughout South America and provide medical care to all of those in need. A few of my hobbies include: playing soccer, singing, and making lame jokes.</p><p><i>Favorite Lab Moment:</i><br />When Greg finally admitted to us being best friends<br />(although some may doubt it ever happened).</p></div>
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<h4>THE QUORUM SENSING SYSTEM OF PSEUDOMONAS AERUGINOSA</h4>
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<p>
 
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The virulence of P. aeruginosa bacteria is in part controlled by their ability to sense each other by producing and recognizing specific compounds called N-acyl homoserine lactones (AHLs), thus allowing them to communicate with each other.</p> <p>This ability to communicate intercellularly, called quorum-sensing, is used by many bacteria to control population growth through gene activation. <figure id="quorumsensing"> <img src="https://static.igem.org/mediawiki/2014/b/b7/Stony_Brook_Project_Quorum_sensing.png"><figcaption> A simple illustration of quorum sensing: Bacteria produce cell-signaling molecules, and at a certain concentration of cells these molecules interact with other bacteria, serving as a communication system </figcaption></figure>As the bacteria grow, they release signaling signals. Once these signals reach a critical concentration, other bacteria in the environment will recognize these signals and respond in turn by making changes in their gene regulation, allowing them to cooperate with each other as their population reaches a certain density. <sup><a href="#13">[6]</a></sup></p>
 
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<p> P. aeruginosa primarily has two quorum-sensing systems: the las system, which consists of the transcriptional regulator LasR and the synthase protein LasI, and the rhl system, which consists of RhlR and RhlI. LasI and RhlI help in the production of the AHL compounds 3-oxo-C12-HSL and C4-HSL respectively. These compounds bind with their respective transcriptional regulators, LasR or RhlR, which then binds to DNA and regulates the transcription of genes. While both 3-oxo-C12-HSL and C4-HSL freely diffuse out of bacterial cells, 3-oxo-C12-HSL diffuses at a much slower rate than C4-HSL. Both of these systems control the pathogenesis of P.aeruginosa, acting as virulence factors as well as communicative signals and other regulatory factors. <sup><a href="#14">[7]</a></sup></p> <figure class="center"><img src="https://static.igem.org/mediawiki/2014/5/53/Stony_Brook_Project_Rhlr_system.png"><figcaption> As the autoinducer rhli is produced, it forms a complex with C4HSL, which then in turn activates the production of regulatory protein rhlr, which then regulates other genes. </figcaption></figure>
 
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<p><h4>EXPLOITING THE QUORUM-SENSING SYSTEM</h4>
 
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By inserting the genes for the rhl sensing system, non-pathogenic i.e. harmless E. coli can be induced to sense P. aeruginosa signals and act in response to them. If a bacteria-killing compound could be produced upon receiving C4HSL or 3-oxo-C6HSL signals, our project could take advantage of naturally-occurring pathogenic bacterial cell communication in order to kill them off. Since C4-HSL diffuses more quickly, we decided to use the rhl system used by P.aeruginosa as a means of identifying our target bacteria. By exploiting the rhl system in P.aeruginosa, we could
 
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target them with an antibiotic-like compound, thus killing our bacteria.
 
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Our next step was to look into our bacteria-killing compound: melittin, a honeybee venom compound.
 
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</p>
 
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<p><h4>
 
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REFERENCES</h4>
 
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<ol>
 
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<a id="8"></a><li> Li XZ, Livermore DM, Nikaido H. 1994. Role of efflux pump(s) in intrinsic resistance of
 
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Pseudomonas aeruginosa: resistance to tetracycline, chloramphenicol, and norfloxacin.
 
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Antimicrobial Agents and Chemotherapy. 38:1732-1741</li>
 
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<a id="9"></a><li> Döring G, Parameswaran IG, Murphy TF. 2011. Differential adaptation of microbial
 
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pathogens to airways of patients with cystic fibrosis and chronic obstructive pulmonary
 
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disease. FEMS Microbiol. Rev. 35:124–146</li>
 
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<a id="10"></a><li> Bodey GP, Bolivar R, Fainstein V, Jadeja L. 1983. Infections caused by Pseudomonas
 
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aeruginosa. Rev Infect Dis. 5:279-313.</li>
 
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<a id="11"></a><li> "Pseudomonas aeruginosa in Healthcare Settings." Centers for Disease Control and
 
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Prevention. Centers for Disease Control and Prevention, 7 May 2014. Web. 3 June 2014.</li>
 
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<a id="12"></a><li> Septimus EJ, Kuper KM. 2009. Clinical challenges in addressing resistance to
 
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antimicrobial drugs in the twenty-first century. Clin Pharmacol Ther. 86(3): 336-339.</li>
 
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<a id="13"></a><li> Difri, CD. 2008. Difri, CD. 2008. Quorum Sensing: Bacteria Talk Sense. Clin Infect Dis. 47 (8): 1070-1076</li>
 
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<a id="14"></a><li> Smith RS, Iglewski BH. 2003. Pseudomonas aeruginosa quorum sensing as a potential
 
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antimicrobial target. J Clin Invest. 112(10): 1460-1465</li></ol></p>  </p>
 
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<h4>HONEYBEE VENOM: PREPROMELITTIN AND MELITTIN</h4>
 
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<p>Honeybees, or Apis mellifera, are native to Europe, Africa, and western Asia. The introduction of Apis mellifera to other parts of the world began in the 17th century and since then, honeybees can be found around the world. Currently, there are 26 recognized subspecies of Apis mellifera based on morphology and molecular differences, as well as the differences in their habitat.While male bee drones do not have stingers, female bees have a stinger, which acts as a form of defense against predation. It is connected to a sac that releases a venom when the bee stings another organism. <figure id="bee"> <img src="https://static.igem.org/mediawiki/2014/e/e4/Stony_Brook_Project_Apismellifera.jpg" height="300" width="400"> <figcaption> Apis mellifera( <a href="http://en.wikipedia.org/wiki/Italian_bee#mediaviewer/File:Honeybee-27527-1.jpg"> Source </a>)  </figcaption></figure></p>
 
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<p>Bee venom has been researched for its ability to treat inflammation, infections, and auto-immune diseases. The active portion of the venom causes inflammation and acts as an anticoagulant. The primary active component of bee venom is melittin, which constitutes about 52% of the apitoxin, or bee venom, liquid excreted from the bee. <sup><a href="#15">[1]</a></sup> An antimicrobial peptide, melittin is a part of the host defense system and immune response of honeybees. Previous research has shown it to be effective with some modifications in both treating cancerous cells and HIV.<sup><a href="#16">[2]</a></sup><sup><a href="#17">[3]</a></sup></p>
 
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<p>Melittin is not produced in bees in its active form. Rather, it is produced in an inactivated form called prepromelittin, which contains the right sequences to send it to the endoplasmic reticulum in bee cells as well as deactivating it.
 
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Sequence prepromelittin
 
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Naturally, bees produce prepromelittin in their venom gland, which gets degraded by surrounding enzymes. <sup> <a href="#18">[4]</a></sup> As the prepromelittin is processed in the honeybee cell, the “pre” part of the peptide, which acts as a signal peptide, is cleaved off by signal peptidase enzymes. <sup> <a href="#19">[5]</a></sup> The “pro” sequence, which acts as a protective sequence which inactivates melittin, is slowly cleaved off stepwise by surrounding dipeptidase enzymes as well, allowing the melittin to become liberated.<sup><a href="#20">[6]</a></sup></p>
 
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<figure class="center"><img src="https://static.igem.org/mediawiki/2014/1/1a/Stony_Brook_Project_Prepromelittin_sequence.png"><figcaption> Sequence of prepromelittin, where green indicates "pre" sequence, red indicated "pro" sequence, and blue indicates the melittin sequence.</figcaption></figure>
 
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<h4>MELITTIN IN ACTION </h4>
 
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<p>Melittin is a small ampipathic peptide made up of 26 amino acids, with a hydrophobic N-terminus and a hydrophilic C-terminus.As melittin concentration in the membrane accumulates, transient pores are formed which allow the permeability of ions. <figure id="melittin"><img src="https://static.igem.org/mediawiki/2014/0/0c/Stony_Brook_Project_Melittin.jpg"><figcaption>Melittin's alpha-helical structure.</figcaption></figure>As the concentration of melittin increases further, pores are stabilized and thus become large enough to allow the movement of large molecules outside the cell, and at even higher concentrations, the membrane will disintegrate entirely. Effectively, the melittin compound will cause cells to lyse either due to osmotic pressure around the cell, or through a detergent-effect at high enough concentrations.
 
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Its activity as a cytolytic compound is due to its surface activity. At low concentrations, it adopts an inactive parallel orientation to the cell membrane, and at higher concentrations it adopts a perpendicular orientation to the membrane, which allows it to “wedge” itself in the headgroup space of phospholipids in a phospholipid bilayer, without extending all the way to the center of the bilayer. As a result, the bilayer distorts and curves to fill in the space left by the “wedge”, forming transient pores through the resulting area imbalance between the inner and outer part of the membrane. Through the transient pores, melittin molecules redistribute, eventually forming larger stable pores made up of 4-7 melittin monomers each.<sup><a href="#21">[7]</a></sup> </p>
 
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<h4>WHY USE MELITTIN?</h4>
 
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<p><figure id="pores"> <img src="https://static.igem.org/mediawiki/2014/7/77/Stony_Brook_Project_Melittin_pore_formation_mechanism.jpg"><figcaption> Mechanism of pore formation (<a href="http://neutron.kaist.ac.kr/research/poreformation.jpg"> Source </a>) </figcaption></figure>Since the majority of antibiotics target parts of bacteria which have mutated to develop resistance, the most effective alternative to antibiotics would work against conserved parts of the bacteria, which cannot mutate easily without compromising its ability to thrive. An example of such an area would be the outermost part of the bacteria, the cell membrane. Melittin can work against pathogenic bacteria by targeting their cell membrane. Thus, melittin can be utilized against pathogenic bacteria as an alternative to antibiotics, as well as a solution to the increasing resistance of bacteria against antibiotics.</p>
 
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<h4> REFERENCES</h4>
 
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<ol><a id="15"></a><li>Meier J, White J. (1995). Clinical toxicology of animal venoms and poisons. CRC Press, Inc. ISBN 0-8493-4489-1.</li>
 
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<a id="16"></a><li>Soman NR, Baldwin SL, Hu G, Marsh JN, Lanza GM, Heuser JE, Arbeit JM, Wickline SA, Schlesinger PH. 2009. Molecularly targeted nanocarriers deliver the cytolytic peptide specifically to tumor cells in mice, reduing cancer growth. J Clin Invest. 199(9): 2830-2842</li>
 
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<a id="17"></a><li>Hood JL, Jallouk AP, Campbell N, Ratner L, Wickline SA. 2013. Cytolytic nanoparticles attenuate HIC-1 infectivity. Antivi Ther. 18(1):95-103</li>
 
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<a id="18"></a><li>Müller G, Zimmermann R. 1987. Import of honeybee prepromelittin into the endoplasmic reticulum: structural basis for independence of SRP and docking protein. EMBO J. 6:2099-2107</li>
 
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<a id="19"></a><li>Mollay C, Vilas U, Kreil G. 1982. Cleavage of honeybee prepromelittin by an endoprotease from rat liver microsomes: identification of intact signal peptide. Proc Natl Acad Sci USA. 79(7): 2260-2263</li>
 
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<a id="20"></a><li>Kreil G, Haiml L, Suchanek G. 1980. Stepwise cleavage of the pro part of promelittin by dipeptidylpeptidase IV. Evidence for a new type of precursor-product conversion. Eur J Biochem. 111(1):49-58 </li>
 
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<a id="21"></a><li>Lee MT, Sun TL, Hung WC, Huang HW. 2013. Process of inducing pores in membranes by melittin. Proc Natl Acad Sci USA. 110(35): 14243-8</li></ol>
 
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<h4> GST-MELITTIN </h4>  <figure class="center"><img src="https://static.igem.org/mediawiki/2014/7/7f/Stony_Brook_Project_GST-Melittin.png"> </figure><p>In order to prevent melittin from either attacking our own cells or from congregating into inactive tetramers, melittin itself cannot be produced within our E.coli cells. However, if we were to produce the naturally-occurring form of prepromelittin, we would also need to recreate the enzymes within the bee. Reproducing these enzymes in an E. coli cell can quickly become complicated, therefore our project uses the GST tag to inactivate melittin, creating a fusion protein. This tag also allows for easy identification by through gel electrophoresis, allowing us to also see if the melittin was produced. A TEV protease site sits in between the GST tag and melittin, allowing for easy cleavage of the GST tag and activation of melittin upon the addition of the TEV protease.</p>
 
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<h4>RHLR BIOSENSOR</h4>
 
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<figure class="center"><img src="https://static.igem.org/mediawiki/2014/6/6b/Stony_Brook_Project_RhlR.png"></figure><p>We designed a biosensor which could detect the quorum sensing signals of P. aeruginosa. This biosensor would constitutively produce the RhlR regulatory protein in order to bind with C4HSL molecules, which then would activate the fluorescence marker mCherry. By responding to C4HSL molecules in the environment, our biosensor could then activate the production of our antibacterial compound, melittin. This form of mechanism allows for a more fine-tuned production of melittin, and acts as a preliminary check for biosafety and the efficiency of our circuit.</p>
 
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<h4>OUR COMPLETE CIRCUIT(RHLR+MELITTIN)</h4>
 
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<figure class="center"><img src="https://static.igem.org/mediawiki/2014/4/47/Stony_Brook_Project_Entire_circuit.png"></figure>
 
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<p> In our complete circuit, our <i>E. coli</i> would constitutively produce RhlR proteins, which then could bind with any C4HSL signal molecules in the environment released by P. aeruginosa. Then, this would activate the <i>rhl</i> promoter, starting the production of GST-melittin. We then would exogenously add in TEV protease, in order to cleave off the GST protein and allow the melittin to work against our target bacteria. Both the RhlR and GST-melittin have fluorescence markers, to allow us to easily monitor the progress of the mechanism. In addition, the use of non-specific melittin also acts as a "kill switch", as our <i>E.coli</i> would eventually succumb to the pores created by melittin. </p>
 
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Home

Team

Project

Notebook

Results

Outreach

Safety

Attributions

Meet the Team!

Our advisors

John Peter Gergen
Department of
Biochemistry and
Cell Biology
Kevin Czaplinski
Department of
Biochemistry and
Cell Biology
Gabor Balazsi
Department of
Biomedical Engineering
Joshua Rest
Department of
Ecology and
Evolution
Ete Chan
Department of
Biomedical Engineering
Yizhi Meng
Department of
Materials Science
and Engineering

Team Members

Gregory Poterewicz

Year: Junior
Major: Biology

When I finally get to leave the lab, I go to sleep for the few hours that I have. When I actually have time, I love to go out into the world and explore, always looking to have new experiences. I am hoping to go to grad school after Stony Brook where my research will conveniently take me to exotic places.

Favorite Lab Moment:
When we found out about our anonymous donor because it meant that the work we were putting in meant something to someone.

Helen Liu

Year: Sophomore
Major: Chemical Engineering

I've often been told I have a surprising sense of humor. Sometimes I even surprise myself. In my free time, I like to watch TV, or sleep, or fall asleep while watching TV. After helping to build the wiki, I'm looking forward to not looking at HTML/CSS for very long time.

Favorite Lab Moment:
When Nic, Gurkamal and I masterminded a kidnapping of Gouda's origami cranes when he went on vacation. (But we all know the real mastermind was me.)

Janki Patel

Year: Sophomore
Major: Biochemistry

One day I plan to one day attend medical school and eventually achieve a career as a doctor. After shadowing around the medical field for a year, the study of the brain has particularly fascinated me, so neurology is a field that I'm looking to pursue. While attending Stony Brook, I will further my knowledge in these areas and contribute to my community through both volunteering and research.

Favorite Lab Moment:
The first time I ever touched anything in the lab. A gel crumbled in my hands and I was afraid to do any lab work for a solid month

Maryam Ige

Year: Junior
Major: Biomedical Engineering

I’ve often been told that I am a challenge driven person. I can’t help that I relish the feeling of accomplishment. So I’m often in situations where I am testing my limits. But what THE ultimate challenge to me is, is dabbling in the impossible; having my imagination brought to life. Hence, iGEM was like a blessing for me: it empowered with the opportunity to create biological systems not found in nature. So cool!!!!

Favorite Lab Moment:
When I became a pro in origami. Thanks Helen!

Gurkamal Kaur

Year: Junior
Major: Biology

Hello my name is Gurkamal Kaur! Here at Stony Brook, I am pursuing a career in medicine and hope to become a future educator in the medical field. Every summer, I participate in a summer day camp and teach elementary school children reading and math skills. It is quite satisfying to know you are making a difference in a child's life and inspiring them to learn while having fun! In my free time, I like to engage in long distance running and playing the lovely sport of volleyball! Above all, there is nothing quite like going hiking with my amazing dog, Oreo!

Favorite Lab Moment:
Streaming the FIFA world cup in lab. Talk about multi-tasking!

Karen Wong

Year: Junior
Major: Biology
Minor: Biomedical Engineering

I'm on the Pre-Med track, but after joining the iGEM team, I realized that I should keep an open mind to graduate school or a career in different areas of science and medicine. I am very outspoken about my thoughts on feminism and the equality between men and women, but I am constantly learning new perspectives and ideas that add to my knowledge of the issue. I want boys and girls to grow up with as few differences as possible.

Favorite Lab Moment:
Debating with Greg as to why I don’t like the term “chick flick.”

Mohamed Gouda

Year: Junior
Major: Biochemistry

I hold a number of interests outside of studying and pursuing a career in medicine. High on that list would have to be chess. I enjoy playing, learning, and even watching games of chess. I'm also a fan of watching films; drama, comedy, thriller, you name it, I enjoy it.

Favorite Lab Moment:
That one time I did something so cool Nic ripped his shorts.

Andrew Xu

Year: Sophomore
Major: Biology

My absolute favorite thing to do in the world is to take a nap. I never quite get enough sleep as a pre-medical student. Of course, I still love studying for my science courses because I aspire to practice in the field of sports medicine. In between the long arduous hours at the library, I do enjoy training as a powerlifter in the gym and hunting for my next junk food fix. But, if you see me, I will love you very much if you can spare a cup of black coffee or an energy drink!

Favorite Lab Moment:
That one day Helen didn't hate me.

Nicolai Tayco

Year: Sophomore
Major: Biochemistry
Minor: Studio Art

My dream, aside from continuing research and going to medical school, is to is to pursue my love of art. I initially planned on droppping it after high school so I can focus on my major, but I just couldn't stay away from it. I've heard a lot of arguments for the discontinuation of art education in schools, but the arts and sciences go hand-in-hand more than people realize. I believe in a well-rounded education for future generations.

Favorite Lab Moment:
That one time Gouda did something so cool I ripped my shorts.

Tenzing Lama

Year: Senior
Major: Biochemistry
Minor: Chemistry

Hi! My name is Tenging Lama. After taking several upper level science courses, and working in an organic synthesis laboratory, I have developed a serious knack of learning more sciences, and I am extremely interested in pursuing my own independent research sometime in the future. In my free time, I like to get together with my good friends and travel to new exciting places.

Favorite Lab Moment:
“That’s it!” – Only few would understand this catchphrase!

Millicent Mulieri

Year: Sophomore
Major: Biochemistry
Minor: Political Science

My name is Millicent Mulieri, but I go by Millie. I am an active member of the Global Medical Brigades and plan on joining Doctors Without Borders upon completion of medical school. My ultimate goal is to set up free healthcare clinics throughout South America and provide medical care to all of those in need. A few of my hobbies include: playing soccer, singing, and making lame jokes.

Favorite Lab Moment:
When Greg finally admitted to us being best friends
(although some may doubt it ever happened).

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