Team:Carnegie Mellon

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<a href="https://2012.igem.org/Team:Carnegie_Mellon/Hum-Team">Team Presentation</a>
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<li><a href="#section1-1">1.1 Question 1</a></li>
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<title>Carnegie_Mellon iGEM 2012</title>
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<i><div class="glow-title">Real-time quantitative measurement of RNA and protein levels using fluorogen-activated biosensors </div></i>
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<b> Our primary goal is to develop new promoters that can be measured with fluorescent technology.</b>
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<li><p> We seek to develop a system that will allow researchers in the field of synthetic biology to accurately measure a variety of metrics in gene expression networks including translational efficiency and transcriptional strength.
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<li><p> We hypothesize that we can use Spinach (a fluorogen-activating RNA sequence) and a FAP (fluorogen activating protein) as biosensors to measure these gene expression metrics <i>in vivo</i> (in living cells), rather than <i>in vitro</i> (in a test tube), which can be very costly and labor intensive.
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<h3><b>Experimental</b></h3>
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The design and implementation of synthetic biological systems often require information on transcription and translation rates and on the impact of both RNA and protein levels on metabolic activities of host cells. Such information is needed when both strong and low levels of expression are desired, depending on the biologists’ goal, e.g., high production or single-molecule localization of a protein, respectively. To date, however, quantitative information about the expression strength of a promoter is difficult to obtain due to the lack of noninvasive and quick approaches to measure levels of RNA and protein in cells.
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Here, we engineer a fluorescence-based biosensor that can provide information on both transcription strength and translation efficiency that is noninvasive, easily applied to a variety of promoters, and capable of providing results in a time frame that is short when compared to current technologies.  The sensor is based on the use of an RNA aptamer (termed Spinach) and a fluorogen activating protein (FAP). Both the Spinach and FAP become fluorescent in response to binding with dye molecules. The combination of FAP and Spinach will allow us to quantitatively determine relationships involving mRNA and protein, such as translational efficiency.
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To demonstrate the utility of the sensor, we have constructed and characterized four T7Lac promoters. For each of the promoters, we have measured both mRNA and protein fluorescence over time. The time-lapse fluorescence levels of mRNA and protein were used in a mathematical model for the estimation of transcription and translation rate constants.
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We have submitted these promoters to the parts registry, whose strength is measured by the newly developed biosensor.
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Human Practices
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The impact of synthetic biology depends on the number and quality of scientists making significant contributions to the field. To this end, we contributed to raising the awareness of high school students, who may become future scientists, about the interdisciplinary field of synthetic biology, and about the preparation one needs to become a synthetic biologist.
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We decided to create teaching materials for high school students inspired by our team’s research project. Our goal was that these materials can be easily used by a science teacher in a lecture in a Biology or Chemistry course to (1) explain what Synthetic Biology is, and (2) enable the students to understand how our <a href="https://2012.igem.org/Team:Carnegie_Mellon/Met-Overview"> biosensor</a> works. The teaching materials we have created, specifically a power point presentation and an <a href="https://2012.igem.org/Team:Carnegie_Mellon/Hum-Circuit"> electronic circuit kit</a>, have become part of the Lending Library of Kits of <a href="http://www.cmu.edu/cnast/DNAZone/index"> DNAZone</a>, the outreach program of the <a href="http://www.cmu.edu/cnast/"> Center of Nucleic Acids Science and Technology (CNAST)</a> at Carnegie Mellon. The kits in the Library are loaned to high school teachers in the Pittsburgh area to be used in teaching Math and Science. We have also tested the kit in several demonstrations in the Summer of 2012 to high school students enrolled in the <a href="http://www.cmu.edu/enrollment/summerprogramsfordiversity/sams.html"> Summer Academy of Math and Science (SAMS)</a> at Carnegie Mellon.
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To bridge the gap between the background of a high school student and the complexity of our project, we built an affordable, microcontroller-based, <a href="https://2012.igem.org/Team:Carnegie_Mellon/Hum-Circuit"> hardware platform</a> and associated, open-source, digital simulation <a href="https://2012.igem.org/Team:Carnegie_Mellon/Hum-Software"> software</a>. The combined hardware/software platform allows the students to directly manipulate electronic components, which are formal equivalents of the BioBricks used in our sensor, and to observe the effect of changing these components on the current or voltage output, which is the equivalent of the fluorescence intensity in our lab experiments. The software part of the platform includes the same model we created for the analysis of the sensor, and the GUI that facilitates the manipulation of the circuit kit.
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<i><a href="https://2012.igem.org/Team:Carnegie_Mellon/Hum-Overview"> Learn more here</a></i>
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<a name="Objective_1:_A_New_Set_of_Well-Characterized_Promoters"></a><h1> <span class="mw-headline"> Objective 1: Novel Well-Characterized Promoters </span></h1>
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<div class="thumb tright"><div class="thumbinner" style="width:152px;"><a href="/Image:Carnegie_Mellon-MicroMaize.jpg" class="image" title="Fluorescence Microscopy"><img alt="Fluorescence Mircroscopy" src="/wiki/images/thumb/0/08/Carnegie_Mellon-MicroMaize.jpg/150px-Carnegie_Mellon-MicroMaize.jpg" width="150" height="301" border="0" class="thumbimage" /></a>  <div class="thumbcaption"><div class="magnify"><a href="/Image:Carnegie_Mellon-MicroMaize.jpg" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>
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Fluorescence Microscopy</div></div></div><font size="4">
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<p><b>Our first objective is to develop a series of BioBricks that are well characterized based on our methods of measurement.</b></p></font>
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<p>We assert that our new method of analyzing promoters can quantify certain properties such as: </p>
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<ol><li> Translational efficiency <i>in vivo </i>
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</li><li> <i>In vivo</i> transcription rates
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</li><li> Promoter strength
 +
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 +
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<p>The promoters we submit were characterized with these properties. </p>
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<strong>Figure 1: Measured transcription (left panel) and translation (right panel) rate constants of three new promoters using a new fluorogen-activated biosensor. </strong>
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<br>  Based on established parts, we have developed a new biosensor that can report levels of both RNA and protein in a single cell. This biosensor enables non-invasive and real-time measurements of RNA and protein expression rates. We have applied the biosensor in the characterization of three new T7Lac promoters, which yielded high quality time-lapse data of both RNA and protein levels (see details in <a href = "https://2012.igem.org/Team:Carnegie_Mellon/Met-Overview"> Methods & Results </a>). The data was used to estimate transcription and translation rate constants (see details in <a href =" https://2012.igem.org/Team:Carnegie_Mellon/Mod-Overview"> Modeling </a>).
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<a name="Objective_2:_Human_Practices"></a><h1> <span class="mw-headline"> Objective 2: Human Practices</span></h1>
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<p> As part of our project, we seek to intrigue high school students about synthetic biology and engineering. In this pursuit, we developed an electrical analog of our BioBricks (with a simulated microscope using LEDs and a photoresistor) to teach high school students about:
-
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+
</li><li> How synthetic biologists tackle real-world problems
-
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+
</li><li> The iGEM competition and how our iGEM team's project enables one to measures the properties of promoters
-
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+
</li><li> The interdisciplinary nature of synthetic biology
 +
</li><li> The advantages and challenges of interdisciplinary work
 +
</li></ol>
 +
</p>
 +
<br \><br \>
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<ul>
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<li><a href="https://2012.igem.org/Team:Carnegie_Mellon" title="Home"><span>Home</span></a></li>
+
-
<li><a href="https://2012.igem.org/Team:Carnegie_Mellon/Plan" title="Plan of the biology" rel="dropmenu1_a"><span>Plan</span></a></li>
+
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<li><a href="https://2012.igem.org/Team:Carnegie_Mellon/Protocols" title="New Protocols developed" rel="dropmenu1_a"><span>Protocols</span></a></li>
+
-
<li><a href="https://2012.igem.org/Team:Carnegie_Mellon/Notebook" title="Details of labwork, in realtime"><span>Notebook</span></a></li>
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+
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+
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+
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+
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<li><a href="https://2012.igem.org/Team:Carnegie_Mellon/ELSI" title="Ethical, legal and social implications of our project" ><span>ELSI</span></a></li>
+
-
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+
-
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+
-
<li><a href="https://2012.igem.org/Team:Carnegie_Mellon/Acknowledgements" title="Acknowledgements" ><span>Acknowledgements</span></a></li>
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<a href="https://2012.igem.org/Team:Carnegie_Mellon/Team">Overview</a>
+
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+
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<p><br />
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<i><b>Welcome to the University of Carnegie_Mellon 2012 iGEM Team Wiki!</b></i>
+
-
<br /><br />
+
-
</p><p><a href="/Image:Carnegie_Mellon-Panorama.jpg" class="image" title="Image:Carnegie_Mellon-Panorama.jpg"><img alt="Image:Carnegie_Mellon-Panorama.jpg" src="/wiki/images/7/7a/Carnegie_Mellon-Panorama.jpg" width="967" height="229" border="0" /></a>
+
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+
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<hr />
+
-
<table id="toc" class="toc" summary="Contents"><tr><td><div id="toctitle"><h2>Contents</h2></div>
+
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+
-
<li class="toclevel-1"><a href="#Introduction:_Global_problems"><span class="tocnumber">1</span> <span class="toctext">Introduction: Global problems</span></a></li>
+
-
<li class="toclevel-1"><a href="#Primary_Objective:_A_single_sustainable_bacterial_solution"><span class="tocnumber">2</span> <span class="toctext">Primary Objective: A single sustainable bacterial solution</span></a></li>
+
-
<li class="toclevel-1"><a href="#Secondary_Objective:_Solving_vitamin_A_deficiency_in_the_developing_world"><span class="tocnumber">3</span> <span class="toctext">Secondary Objective: Solving vitamin A deficiency in the developing world</span></a></li>
+
-
<li class="toclevel-1"><a href="#Further_Considerations"><span class="tocnumber">4</span> <span class="toctext">Further Considerations</span></a></li>
+
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+
-
<a name="Introduction:_Global_problems"></a><h2> <span class="mw-headline"> Introduction: Global problems </span></h2>
+
-
<ul><li> It has been predicted that by 2015 supplies of easy-to-access oil and natural gas will no longer keep up with demand. This prediction is based on the current rate of consumption, but this is expect to increase. The repercussions of this are already being felt on a global level with food prices rising.
+
</li></ul>
</li></ul>
-
<ul><li> The burning of fossil fuels is also fuelling global warming. This is having negative effects on crop yields worldwide, with longer droughts occurring year on year, especially in Africa and Australia. The melting of the polar ice-caps which accompanies this will raise sea-levels, inundating currently arable land, reducing the land available for an ever enlarging world population.
+
Advisors for the team are from the Chemistry, Biomedical Engineering, Electrical and Computer Engineering, Computational Biology, and Biological Science Departments.  
-
</li></ul>
+
-
<ul><li> At present the major alternative to fossil fuel use for transport comes from ethanol fermented from starch and sugar in sugar cane and soy bean. These biofuel crops are being grown in areas previously used for food crops or in previously pristine natural environments, and thus are unsustainable.<br />
+
-
</li></ul>
+
-
<p><br />
+
-
All this is burdening the global economy and destroying lives, and worse is likely to come.<br />
+
-
<br />
+
-
<b>Wouldn't it be brilliant if we could do something to counter this trend?</b><br />
+
-
<br />
+
-
This is what the Carnegie_Mellon 2012 iGEM team have been trying to do. - The rising cost of food coupled to the current unsustainability of human activity makes this the perfect time to contemplate the restructuring of global agriculture.<br />
+
</p>
</p>
-
<a name="Primary_Objective:_A_single_sustainable_bacterial_solution"></a><h2> <span class="mw-headline"> Primary Objective: A single sustainable bacterial solution </span></h2>
+
<br \>
-
<div class="thumb tright"><div class="thumbinner" style="width:152px;"><a href="/Image:Carnegie_Mellon-MicroMaize.jpg" class="image" title="A false-colour SEM of MicroMaize cells."><img alt="A false-colour SEM of MicroMaize cells." src="/wiki/images/thumb/0/08/Carnegie_Mellon-MicroMaize.jpg/150px-Carnegie_Mellon-MicroMaize.jpg" width="150" height="301" border="0" class="thumbimage" /></a>  <div class="thumbcaption"><div class="magnify"><a href="/Image:Carnegie_Mellon-MicroMaize.jpg" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>A false-colour SEM of MicroMaize cells.</div></div></div>
+
-
<p>We have been investigating engineering bacteria to <b>produce starch from the cellulose in waste biomass</b> (that is agricultural waste, wood chippings, waste from paper production etc.). This starch could be:
+
-
</p>
+
-
<ol><li> sold to the biofuels industry for conversion to ethanol
+
-
</li><li> used as feed for livestock
+
-
</li><li> used as a starch supplement in the human diet if needed.
+
-
</li></ol>
+
-
<p>This will reduce our dependence on fossil fuels and free up agricultural land for the growth of food crops, ultimately putting less strain on ecosystems.
+
-
</p>
+
-
<a name="Secondary_Objective:_Solving_vitamin_A_deficiency_in_the_developing_world"></a><h2> <span class="mw-headline"> Secondary Objective: Solving vitamin A deficiency in the developing world </span></h2>
+
-
<p>We have also been continuing the work of <a href="https://2007.igem.org/Carnegie_Mellon/Yoghurt" class="external text" title="https://2007.igem.org/Carnegie_Mellon/Yoghurt" rel="nofollow">our 2007 team</a> in engineering <i>Escherichia coli</i> to <b>produce the vitamin A precursor β-carotene</b>. Vitamin A is required for vision and a healthy immune system. 250,000-500,000 children in the developing world lose their vision each year, half of them dying within 12 months of this (<a href="http://www.who.int/nutrition/topics/vad/en/" class="external text" title="http://www.who.int/nutrition/topics/vad/en/" rel="nofollow">WHO</a>).
+
-
</p>
+
-
<a name="Further_Considerations"></a><h2> <span class="mw-headline"> Further Considerations </span></h2>
+
-
<p>In the pursuit of our project, as well as the biological aspects, we:
+
-
</p>
+
-
<ul><li> considered aspects of <a href="https://2012.igem.org/Team:Carnegie_Mellon/Modelling" class="external text" title="https://2012.igem.org/Team:Carnegie_Mellon/Modelling" rel="nofollow">scale-up</a>, including the <a href="https://2012.igem.org/Team:Carnegie_Mellon/ELSI" class="external text" title="https://2012.igem.org/Team:Carnegie_Mellon/ELSI" rel="nofollow">ethical, legal and social implications</a> of our potential final product, MicroMaize,
+
-
</li><li> programmed <a href="https://2012.igem.org/Team:Carnegie_Mellon/Software" class="external text" title="https://2012.igem.org/Team:Carnegie_Mellon/Software" rel="nofollow">a new piece of software</a> for use in metabolic modelling,
+
-
</li><li> <a href="https://2012.igem.org/Team:Carnegie_Mellon/Protocols" class="external text" title="https://2012.igem.org/Team:Carnegie_Mellon/Protocols" rel="nofollow">developed and tested a number of new techniques</a> to make the process of creating BioBricks<sup>TM</sup> that little bit easier,
+
-
</li><li> collaborated with <a href="https://2012.igem.org/Team:Guelph" class="external text" title="https://2012.igem.org/Team:Guelph" rel="nofollow">the University of Guelph team</a>, sharing carotenoid synthesis genes, flux modulators, and gram positive plasmids.
+
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</li></ul>
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<a name="Further_Considerations"></a><h1> <span class="mw-headline"> Further Considerations </span></h1>
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<p>In the pursuit of our project we:
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<ul><li> Considered the <a href="https://2012.igem.org/Team:Carnegie_Mellon/Hum-Overview" rel="nofollow">ethical, legal and social implications</a> of our BioBrick
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</li><li> Developed and tested <a href="https://2012.igem.org/Team:Carnegie_Mellon/Met-Protocols" rel="nofollow">techniques for measuring translational efficiency and transcriptional strength </a>
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</li><li> Created materials for teaching high school students about synthetic biology and scientific research.  These materials included a programmable and interactive, <a href="https://2012.igem.org/Team:Carnegie_Mellon/Hum-Circuit" rel="nofollow">electrical analog of our biosensor.</a>
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Latest revision as of 03:26, 27 October 2012

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Real-time quantitative measurement of RNA and protein levels using fluorogen-activated biosensors



Introduction: Motivation and Background

Our primary goal is to develop new promoters that can be measured with fluorescent technology.

  • We seek to develop a system that will allow researchers in the field of synthetic biology to accurately measure a variety of metrics in gene expression networks including translational efficiency and transcriptional strength.

  • We hypothesize that we can use Spinach (a fluorogen-activating RNA sequence) and a FAP (fluorogen activating protein) as biosensors to measure these gene expression metrics in vivo (in living cells), rather than in vitro (in a test tube), which can be very costly and labor intensive.

  • We aim to characterize the relationship between synthesis rates of Spinach and transcription rates and the relationship between synthesis rates of FAP and translation rates.



    • Project Description

    Experimental

    The design and implementation of synthetic biological systems often require information on transcription and translation rates and on the impact of both RNA and protein levels on metabolic activities of host cells. Such information is needed when both strong and low levels of expression are desired, depending on the biologists’ goal, e.g., high production or single-molecule localization of a protein, respectively. To date, however, quantitative information about the expression strength of a promoter is difficult to obtain due to the lack of noninvasive and quick approaches to measure levels of RNA and protein in cells.

    Here, we engineer a fluorescence-based biosensor that can provide information on both transcription strength and translation efficiency that is noninvasive, easily applied to a variety of promoters, and capable of providing results in a time frame that is short when compared to current technologies. The sensor is based on the use of an RNA aptamer (termed Spinach) and a fluorogen activating protein (FAP). Both the Spinach and FAP become fluorescent in response to binding with dye molecules. The combination of FAP and Spinach will allow us to quantitatively determine relationships involving mRNA and protein, such as translational efficiency.

    To demonstrate the utility of the sensor, we have constructed and characterized four T7Lac promoters. For each of the promoters, we have measured both mRNA and protein fluorescence over time. The time-lapse fluorescence levels of mRNA and protein were used in a mathematical model for the estimation of transcription and translation rate constants. We have submitted these promoters to the parts registry, whose strength is measured by the newly developed biosensor.

    Learn more here

    Human Practices

    The impact of synthetic biology depends on the number and quality of scientists making significant contributions to the field. To this end, we contributed to raising the awareness of high school students, who may become future scientists, about the interdisciplinary field of synthetic biology, and about the preparation one needs to become a synthetic biologist.

    We decided to create teaching materials for high school students inspired by our team’s research project. Our goal was that these materials can be easily used by a science teacher in a lecture in a Biology or Chemistry course to (1) explain what Synthetic Biology is, and (2) enable the students to understand how our biosensor works. The teaching materials we have created, specifically a power point presentation and an electronic circuit kit, have become part of the Lending Library of Kits of DNAZone, the outreach program of the Center of Nucleic Acids Science and Technology (CNAST) at Carnegie Mellon. The kits in the Library are loaned to high school teachers in the Pittsburgh area to be used in teaching Math and Science. We have also tested the kit in several demonstrations in the Summer of 2012 to high school students enrolled in the Summer Academy of Math and Science (SAMS) at Carnegie Mellon.

    To bridge the gap between the background of a high school student and the complexity of our project, we built an affordable, microcontroller-based, hardware platform and associated, open-source, digital simulation software. The combined hardware/software platform allows the students to directly manipulate electronic components, which are formal equivalents of the BioBricks used in our sensor, and to observe the effect of changing these components on the current or voltage output, which is the equivalent of the fluorescence intensity in our lab experiments. The software part of the platform includes the same model we created for the analysis of the sensor, and the GUI that facilitates the manipulation of the circuit kit.

    Learn more here



    Objective 1: Novel Well-Characterized Promoters

    Our first objective is to develop a series of BioBricks that are well characterized based on our methods of measurement.

    We assert that our new method of analyzing promoters can quantify certain properties such as:

    1. Translational efficiency in vivo
    2. In vivo transcription rates
    3. Promoter strength
    4. In vivo mRNA and protein half-lives in real time


    The promoters we submit were characterized with these properties.


    Figure 1: Measured transcription (left panel) and translation (right panel) rate constants of three new promoters using a new fluorogen-activated biosensor.
    Based on established parts, we have developed a new biosensor that can report levels of both RNA and protein in a single cell. This biosensor enables non-invasive and real-time measurements of RNA and protein expression rates. We have applied the biosensor in the characterization of three new T7Lac promoters, which yielded high quality time-lapse data of both RNA and protein levels (see details in Methods & Results ). The data was used to estimate transcription and translation rate constants (see details in Modeling ).



    Objective 2: Human Practices

    As part of our project, we seek to intrigue high school students about synthetic biology and engineering. In this pursuit, we developed an electrical analog of our BioBricks (with a simulated microscope using LEDs and a photoresistor) to teach high school students about:

    1. Synthetic biology and its relationship to biology, science, and engineering in general
    2. Gene expression and the central dogma of molecular biology
    3. How synthetic biologists tackle real-world problems
    4. The iGEM competition and how our iGEM team's project enables one to measures the properties of promoters
    5. The interdisciplinary nature of synthetic biology
    6. The advantages and challenges of interdisciplinary work



    Further Considerations

    In the pursuit of our project we:

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