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| | #home { | | #home { |
| − | background: url(https://static.igem.org/mediawiki/2015/6/66/HumanPracticeBG.png) 50% 0 no-repeat fixed; | + | background: url(https://static.igem.org/mediawiki/2015/8/87/ElanVitalKorea_bg_3.png) 50% 0 no-repeat fixed; |
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| | #about { | | #about { |
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| | </style> | | </style> |
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| | <br><br><br><br><br><br><br> | | <br><br><br><br><br><br><br> |
| | <div class="center;"> | | <div class="center;"> |
| − | <a href="https://2015.igem.org/Team:elan_vital_korea"> | + | <a href="https://2015.igem.org/Team:Elan_Vital_Korea"> |
| | <img class="displayed" src="https://static.igem.org/mediawiki/2015/d/d5/ElanVital_Logo.png"> | | <img class="displayed" src="https://static.igem.org/mediawiki/2015/d/d5/ElanVital_Logo.png"> |
| | </a> | | </a> |
| | <h4 style="text-align:center;"> | | <h4 style="text-align:center;"> |
| | + | <font color="white"> |
| | PROJECT <br> -Project Overview- | | PROJECT <br> -Project Overview- |
| | </h4> | | </h4> |
| − | <a href="#myAnchor" rel="" id="anchor1" class="anchorLink"><img class="displayed" src="https://static.igem.org/mediawiki/2015/d/d2/Scroll_arrow.png"></a> | + | </font> |
| | + | <a href="#myAnchor" rel="" id="anchor1" class="anchorLink"><img class="displayed" src="https://static.igem.org/mediawiki/2015/d/d2/Scroll_arrow.png"></a> |
| | </div> | | </div> |
| | </section> | | </section> |
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| | <h5 style="text-align:center;"> | | <h5 style="text-align:center;"> |
| | <a name="myAnchor" id="myAnchor"></a> | | <a name="myAnchor" id="myAnchor"></a> |
| − | <br><br> | + | <br><br><br><br><br><br><br><br> |
| − | <font color="white">PROJECT OVERVIEW</font> | + | <font color="black">PROJECT OVERVIEW</font> |
| | </h5> | | </h5> |
| | <br><br> | | <br><br> |
| − | <P style="text-align:center;"> | + | <div class="inner"> |
| − | <font color="white">
| + | <P style="text-align:left;"> |
| − | Bacteria acquiring resistance to antibiotics pose serious health problem globally. Following last year’s example, <br>
| + | <font color="black"> |
| − | the project of Elan Vital Korea for this year also is related to MRSA. This year, however, we have focused <br>
| + | Antibiotic-resistant bacteria pose a serious problem for global medical community. Detecting antibiotic resistance as quickly as possible is crucial for determination of the correct treatment for patients and for setting up quarantines to prevent spreading. We hypothesized that it is possible to use quorum sensing (QS) to devise a rapid way for cells to report the existence of antibiotic-resistant bacteria. |
| − | on early detection of MRSA infection using quorum sensing. Below, we have briefly described the health threats <br>
| + | |
| − | caused by MRSA, and have explained the quorum sensing method. Then, we have proceeded to the description <br>
| + | |
| − | of how we designed and implemented our experiments, and what results we have obtained. Finally, we have briefly <br> | + | |
| − | outlined the implication of our results and future plans. <br><br><br>
| + | |
| | </font> | | </font> |
| | </p> | | </p> |
| − | <img class="displayed" src="https://static.igem.org/mediawiki/2015/6/6e/Hr_white.jpg" width="80px" height="2px">
| + | </div> |
| | <br><br> | | <br><br> |
| | </h6> | | </h6> |
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| − | <h5 style="text-align:center;">
| + | <div class="inner"> |
| − | <font color="white">
| + | <P style="text-align:left;"> |
| − | Threats of Antibiotics-Resistant Bacteria
| + | <font color="black"> |
| − | </h5>
| + | Here, we developed a reporter cell that expresses GFP in the presence of the QS signaling molecule acyl homoserine lactone (AHL). Our test cells (which act as a simulation of antibiotic-resistant bacteria) express lactonase, which breaks down AHL. In our experimental system, test cells should signify their presence by breaking down AHL and preventing GFP expression in reporter cells. Therefore, our project serves as a proof of principle and we hope that our work will serve as a basis for developing similar, more sophisticated quorum sensing-based detection systems for antibiotic-resistant bacteria in the future. |
| − | <br><br>
| + | |
| − | <P style="text-align:center;"> | + | |
| − | Infection by antibiotic-resistant bacteria is a serious health threat worldwide including Korea and <br>
| + | |
| − | the United States of America. It is a serious threat primarily because, as the name suggests, <br> | + | |
| − | bacteria have evolutionarily developed a resistance to antibiotics. It means, first of all, drugs don’t work.<br>
| + | |
| − | Furthermore, the spread of the antibiotic-resistant bacteria makes it more difficult to <br>
| + | |
| − | control or contain the spread of the infectious disease, because it undermines the effectiveness of treatment.<br>
| + | |
| − | And, it substantially increases the cost of healthcare, and the burden to society because it prolongs <br>
| + | |
| − | the treatment period and increases the likelihood of death. WHO declared that it “threatens the achievements of <br>
| + | |
| − | modern medicine” (Antimicrobial Resistance: Global Report on Surveillance 2014, WHO, 2014). <br>
| + | |
| − | Antimicrobial resistance already causes 700,000 deaths every year, which number is expected to 10 million annually<br>
| + | |
| − | by 2050 (An international legal framework to address antimicrobial resistance, WHO, 2015).
| + | |
| | </p> | | </p> |
| | + | </div> |
| | </font> | | </font> |
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| − | To The Top
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| − | <P style="text-align:center;">
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| − | What makes the problem more pressing is that the data isbased on the reports of clinical samples from <br>
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| − | laboratories, “predominantly in hospital settings” (Antimicrobial Resistance: Global Report on <br>
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| − | Surveillance 2014, WHO, 2014, p. 70), which means community-acquired (compared to health-care associated)<br>
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| − | infections and uncomplicated infections are underrepresented. <br><br>
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| − |
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| − | Global Report on Surveillance 2014, WHO, 2014, p. 70), which means community-acquired (compared to health-care <br>
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| − | associated) infections and uncomplicated infections are underrepresented.
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| − | <h5 style="text-align:center;">
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| − | </a><font color="black">Existing Methods Used for Detection</font></h5> <br><br>
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| − | <P style="text-align:center;">
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| − | <font color="black">
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| − | <P style="text-align:center;">
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| − | CDC’s efforts at outsmarting the antibiotic resistance focuses on 4 core actions: detect, respond, prevent <br>
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| − | and discover. The project is called AR Initiative (Detect and Protect Against Antibiotic <br>
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| − | Resistance Initiative), which is an integral part of the CDC strategy to target investment aimed at AR. <br>
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| − | Among the AR initiative, detection is the first step that impacts the whole controlling process. <br>
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| − | Detecting antibiotic resistance quickly and effectively is crucial for determination of the treatment methods<br>
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| − | for different patients as well as for quarantines to prevent it from becoming epidemic. <br>
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| − | Currently, several methods are used for the detection of the antibiotic resistance. Most common and traditional <br>
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| − | method is using growth inhibition assays performed in broth or by agar disc diffusion. <br>
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| − | For clinically critical bacteria, diagnostic laboratories perform phenotypic-based analyses using standardized <br>
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| − | susceptibility testing methods, usually in accordance with the guidelines published by the Clinical <br>
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| − | and Laboratory Standards Institute. <br><br>
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| − | Using the culture-based approach, it can take 1—2 days to produce results for fast-growing bacteria such as <br>
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| − | Escherichia coli orSalmonella, but several weeks for slow-growing bacteria such as Mycobacterium tuberculosis. <br>
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| − | Moreover, culturing only works for a small fraction of microbes; although most pathogens can be cultured <br>
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| − | relatively easily thanks to years of accumulated experimental experiences, the vast majority of microbes cannot <br>
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| − | grow outside their host environment, including pathogens such as Chlamydia orTrypanosomes. <br><br>
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| − | </font>
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| − | </p>
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| − | <a href="#top" rel="" id="top" class="anchorLink">
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| − | <h6 style="text-align:center;">
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| − | To The Top
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| − | </font>
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| − | </h6>
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| − | <br><br>
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| − | <P style="text-align:center;">
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| − | <font color="white">
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| − | Using newer molecular detection techniques for antibiotic resistance such as quantitative PCR (qPCR) or microarrays, we can determine <br>
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| − | the presence of specific resistance genes within hours, and we obtain improved diagnosis results. However, <br>
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| − | these culture-independent approaches target well-known and well-studied pathogens or resistance-causing genes only,<br>
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| − | and cannot be easily used for broader spectrum screening. <br><br>
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| − | </font>
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| − | </p>
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| − |
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| − |
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| − | <P style="text-align:center;">
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| − | <font color="white">
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| − | CDC dramatically innovated the detection process by adopting the Advanced Molecular Detection (AMD), which combines the latest <br>
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| − | pathogen identification technologies with bioinformatics and advanced epidemiology to more effectively understand, prevent and <br>
| |
| − | control infectious diseases. Using those technologies, it is possible to rapidly look for a microbe's match among <br>
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| − | thousands of reference samples in the microbe library. If no match is found, the whole genomic sequence <br>
| |
| − | of the microbe's DNA code can be taken, then quickly analyzed using disease detective works and bioinformatics <br>
| |
| − | to answer critical disease-response questions. However, this new method, while it sounds <br>
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| − | very interesting, is not to be completed until 2020, and still requires incubation, as well as being expensive. <br><br>
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| − |
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| − | </font>
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| − | </p>
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| − | <img class="displayed" src="https://static.igem.org/mediawiki/2015/6/6e/Hr_white.jpg" width="80px" height="2px">
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| − | <br><br>
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| − | <h5 style="text-align:center;">
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| − | <font color="white">
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| − | Our Hypothesis: Possibility of Using Quorum <br>
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| − | Sensing for Early Detection
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| − | </font>
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| − | </h5>
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| − | <br>
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| − | <P style="text-align:center;">
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| − | <font color="white">
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| − | Our team, Elan Vital Korea, addressed the problem of rapidly detecting antibiotic-resistant bacteria. We were interested in <br>
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| − | a rapid and efficient method of antibiotic resistance detection, and we believed that such a method could be engineered <br>
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| − | using quorum sensing. Our hypothesis was that we would be able to use quorum sensing – a method bacteria <br>
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| − | use to communicate with each other – to make the cells quickly report the existence of antibiotic-resistant bacteria
| |
| − | <br><br>
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| − | Using the culture-based approach, it can take 1—2 days to produce results for fast-growing bacteria such as Escherichia coli <br>
| |
| − | orSalmonella, but several weeks for slow-growing bacteria such as Mycobacterium tuberculosis. Moreover, culturing only works <br>
| |
| − | for a small fraction of microbes; although most pathogens can be cultured relatively easily thanks to <br>
| |
| − | years of accumulated experimental experiences, the vast majority of microbes cannot grow outside their host <br>
| |
| − | environment, including pathogens such as Chlamydia orTrypanosomes. <br><br>
| |
| − | </font>
| |
| − | </p>
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| − |
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| − | <a href="#top" rel="" id="top" class="anchorLink">
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| − | <img class="displayed" src="https://static.igem.org/mediawiki/2015/b/b3/Scroll_arrow_top.PNG"></a>
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| − | <h6 style="text-align:center;">
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| − | <font color="white">
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| − | To The Top
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| − | </font>
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| − | </h6>
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| − |
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| − | </section>
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| − | <!-- Section #5 -->
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| − | <section id="maintext" data-speed="10" data-type="background">
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| − | <P style="text-align:center;">
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| − | <font color="black">
| |
| − | <br><br>
| |
| − |
| |
| − | LUsing newer molecular detection techniques for antibiotic resistance such as quantitative PCR (qPCR) or microarrays, we can<br>
| |
| − | determine the presence of specific resistance genes within hours, and we obtain improved diagnosis results. However, these <br>
| |
| − | culture-independent approaches target well-known and well-studied pathogens or resistance-causing genes only,<br>
| |
| − | and cannot be easily used for broader spectrum screening. <br><br>
| |
| − |
| |
| − | CDC dramatically innovated the detection process by adopting the Advanced Molecular Detection (AMD), which combines the latest <br>
| |
| − | pathogen identification technologies with bioinformatics and advanced epidemiology to more effectively <br>
| |
| − | understand, prevent and control infectious diseases. Using those technologies, it is possible to rapidly look for a microbe's match <br>
| |
| − | among thousands of reference samples in the microbe library. If no match is found, the whole genomic sequence of the microbe's <br>
| |
| − | DNA code can be taken, then quickly analyzed using disease detective works and bioinformatics to answer <br>
| |
| − | critical disease-response questions. However, this new method, while it sounds very interesting,<br>
| |
| − | is not to be completed until 2020, and still requires incubation, as well as being expensive. <br><br>
| |
| − |
| |
| − | For the project, we have developed a reporter cell that expresses GFP in the presence of the QS signaling molecule acyl homoserine <br>
| |
| − | lactone (AHL). Our test cells (which act as a simulation of antibiotic-resistant bacteria) express lactonase, which breaks down AHL.<br>
| |
| − | In our experimental system, test cells should signify their presence by breaking down AHL and <br>
| |
| − | preventing GFP expression in reporter cells.
| |
| − |
| |
| − | </font>
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| − | </p>
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| − |
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| − | <br><br>
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| − | <img class="displayed" src="https://static.igem.org/mediawiki/2015/6/6e/Hr_black.jpg" width="80px" height="2px">
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| − | <br><br>
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| − | <h5 style="text-align:center;">
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| − | <font color="black">
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| − | Experiment: Process and Results<br>
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| − | </font>
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| − | </h5>
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| − | <br>
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| − |
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| − | <P style="text-align:center;">
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| − | <font color="black">
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| − | There are many ways of utilizing quorum sensing for medicinal use, and one of the most intuitive and <br>
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| − | most well-known methods is quorum quenching. Quorum quenching takes advantage of the fact that quorum sensing <br>
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| − | also plays a role in expressing virulence, and interferes with the quorum sensing that produces virulence. There are many ways of <br>
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| − | utilizing quorum sensing for medicinal use, and one of the most intuitive and most well-known methods is quorum quenching. <br>
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| − | Quorum quenching takes advantage of the fact that quorum sensing also plays a role in expressing virulence, and<br> interferes with the quorum sensing that produces virulence.<br>
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| − | </font>
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| − | </p>
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| − |
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| − | <a href="#top" rel="" id="top" class="anchorLink"> <img class="displayed" src="https://static.igem.org/mediawiki/2015/5/5b/Scroll_arrow_top_Black.png"></a>
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| − | <h6 style="text-align:center;"> <font color="black">
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| − | To The Top
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| − | </font> </h6>
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| − | </section>
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| − |
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| − | <!-- Section #6 -->
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| − | <section id="maintext2" data-speed="10" data-type="background">
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| − | <br><br>
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| − | <P style="text-align:center;">
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| − | <font color="white">
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| − | However, for our project this year, we decided to focus on engineering a detection method for antibiotic resistance. For the project, <br>
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| − | we created a test plasmid and a reporter plasmid. We then transformed competent E. coli with the plasmids to produce a <br>
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| − | test cell and a reporter cell. As shown in the picture below, the test cell produces lactonase, which breaks down AHL, a common auto-inducer in <br>
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| − | gram-negative bacteria. And the reporter cell produces GFP (or luciferase) which creates a visible difference that we can detect. <br>
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| − | Both plasmids were engineered using the BioBrick DNA recombination process. With such a set up, it will be possible to detect the presence <br>
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| − | of the test cell, or lactonase.<br><br>
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| − |
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| − | For the confirmation of our hypothesis, we conducted some experiments. Ideally, mixing AHL with the test cell will break down the AHL. And, adding <br>
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| − | the reporter after that will not result in any fluorescence. But, if we do the same process with the control bacteria instead of the test cell, <br>
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| − | there will be fluorescence. As theorized, the control experiments produced fluorescence, but the experiments with the test cell produced no fluorescence. <br><br>
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| − | <h6 style="text-align:center;">
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| − | <font color="white">
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| − | To The Top
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| − | </font>
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| − | </h6>
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| − | </section>
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| − | <br><br>
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| − | <h5 style="text-align:center;">
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| − | <font color="black">
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| − | Expected Benefits
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| − | </font>
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| − | </h5>
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| − | <br><br>
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| − |
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| − | <P style="text-align:center;">
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| − | <font color="black">
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| − | Thanks to bacteria’s ability to make quick and profound changes in gene transcription, quorum sensing can be <br>
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| − | used to detect a low amount of signaling molecules and report their presence quickly. With further <br>
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| − | research and thorough engineering applications, it may be possible to detect other antibiotic-resistant bacteria<br>
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| − | that are unknown until now.<br><br>
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| − |
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| − | If it is proven as valid and effective through sufficient tests, this technique could be disseminated to <br>
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| − | hospitals and clinics to test the presence of antibiotic-resistant bacteria. <br>
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| − | We hope that this technique, if properly adjusted for functional advancement, can detect antibiotic-resistant <br>
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| − | bacteria in a relatively short time with only a small amount of sample secured from the patient. <br>
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| − | This would provide an advantage over the traditional detection methods, culture-based approaches which require <br>
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| − | one or several days of incubation period.<br><br>
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| − |
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| − | Because chemicals involved in species-specific quorum sensing is very specific, it might be possible to <br>
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| − | dramatically resolve the problem of overnight incubation. Because an initial sample from <br>
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| − | a patient is usually contaminated and has only a small concentration of the wanted bacteria, it is often <br>
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| − | impossible to detect any antibiotic-resistance without purification and amplification through overnight <br>
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| − | incubation. But because species-specific quorum sensing involves biochemical that are<br>
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| − | highly specific, and the quorum sensing chemicals are not affected as much by the contamination, the method <br>
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| − | utilizing quorum sensing might be applied with relatively less purification processes. Also, because <br>
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| − | some quorum sensing mechanisms have built in positive feedback, with the right engineering, <br>
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| − | the mechanism could work with only a little amplification process.<br><br>
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| − | More innovative detection methods such as quantitative PCR(qPCR) or microarrays, and advanced molecular <br>
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| − | detection (AMD) are based on accumulated previous data and, thus, render very accurate results, but <br>
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| − | they require complicated procedures and heavy equipment. On the other hand, this quorum sensing-based detection <br>
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| − | method will provide benefits to patients with handy procedure and quicker detection results. We believe quicker <br>
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| − | and easy detection of antibiotic-resistant bacteria will lead to better containment of such <br>
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| − | dangerous bacterial strains.<br><br>
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