Team:Carnegie Mellon

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<a href="https://2012.igem.org/Team:Carnegie_Mellon">Home</a>
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<a href="https://2012.igem.org/Team:Carnegie_Mellon">Overview</a>
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<a href="https://2012.igem.org/Team:Carnegie_Mellon/Hom-Introduction">Introduction</a>
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<a href="https://2012.igem.org/Team:Carnegie_Mellon/Hom-Team">Team</a>
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<a href="https://2012.igem.org/Team:Carnegie_Mellon/Hom-Attributions">Attributions</a>
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<a href="https://2012.igem.org/Team:Carnegie_Mellon/Hom-Acknowledgements">Acknowledgements</a>
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<a href="https://2012.igem.org/Team:Carnegie_Mellon/Bio-Overview">BioBricks</a>
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<a href="https://2012.igem.org/Team:Carnegie_Mellon/Bio-Overview">Overview</a>
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<a href="https://2012.igem.org/Team:Carnegie_Mellon/Bio-Submitted">Submitted Parts</a>
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<a href="https://2012.igem.org/Team:Carnegie_Mellon/Met-Overview">Methods and Results</a>
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<a href="https://2012.igem.org/Team:Carnegie_Mellon/Met-Overview">Overview</a>
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<a href="https://2012.igem.org/Team:Carnegie_Mellon/Met-Results">Results</a>
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<a href="https://2012.igem.org/Team:Carnegie_Mellon/Met-Protocols">Protocols</a>
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<a href="https://2012.igem.org/Team:Carnegie_Mellon/Met-Challenges">Challenges</a>
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<a href="https://2012.igem.org/Team:Carnegie_Mellon/Mod-Overview">Modeling</a>
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<a href="https://2012.igem.org/Team:Carnegie_Mellon/Mod-Overview">Overview</a>
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<a href="https://2012.igem.org/Team:Carnegie_Mellon/Mod-Derivations">Derivations</a>
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<a href="https://2012.igem.org/Team:Carnegie_Mellon/Mod-Matlab">Matlab</a>
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<a href="https://2012.igem.org/Team:Carnegie_Mellon/Hum-Overview">Human Practices</a>
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<a href="https://2012.igem.org/Team:Carnegie_Mellon/Hum-Overview">Overview</a>
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<a href="https://2012.igem.org/Team:Carnegie_Mellon/Hum-Outreach">Outreach</a>
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<a href="https://2012.igem.org/Team:Carnegie_Mellon/Hum-Circuit">Circuit Kit</a>
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<a href="https://2012.igem.org/Team:Carnegie_Mellon/Hum-Software">Software</a>
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<a href="https://2012.igem.org/Team:Carnegie_Mellon/Hum-Team">Team Presentation</a>
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<a href="https://2012.igem.org/Team:Carnegie_Mellon/Hum-Teaching">Teaching Presentation</a>
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<li class="toc-h1"><a href="#section1">1. FAQ</a>
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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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<h1> Introduction: Motivation and Background </h1>
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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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<li><p> 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.  
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<a name="Project_description"></a>Project Description</h1>
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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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<i><a href="https://2012.igem.org/Team:Carnegie_Mellon/Met-Overview"> Learn more here</a></i>
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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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<br \><br \>
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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>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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<p>The promoters we submit were characterized with these properties. </p>
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<img src="https://static.igem.org/mediawiki/2012/9/9b/Tl.png" height="300" width="380" >
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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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<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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<li><a href="https://2012.igem.org/Team:Carnegie_Mellon" title="Home"><span>Home</span></a></li>
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<li><a href="https://2012.igem.org/Team:Carnegie_Mellon/Overview" title="Overview of the biology" rel="dropmenu1_a"><span>Overview</span></a></li>
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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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<a href="https://2012.igem.org/Team:Carnegie_Mellon/Team">Step1</a>
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<a href="https://2012.igem.org/Team:Carnegie_Mellon/Team">Step2</a>
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<li class="toclevel-1"><a href="#Primary_Objective:_A_Useful_BioBrick_for_Synthetic_Biologists"><span class="tocnumber">2</span> <span class="toctext">Primary Objective: A Useful BioBrick for Synthetic Biologists</span></a></li>
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<a name="Introduction:_Motivation"></a><h2> <span class="mw-headline"> Introduction: Motivation </span></h2>
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<ul><li> We seek to develop a BioBrick 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></ul>
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<ul><li> We believe that we can use Spinach (a fluorescent RNA sequence) and a FAP (fluorogen activating protein) as biosensors to reflect these metrics <i>in vivo</i>, rather than <i>in vitro</i>, which has previously proven to be very costly and impractical.
 
-
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<ul><li> We will characterize the relationship between genetic expression of Spinach (upstream), a FAP (downstream), translational efficiency, and transcriptional strength.<br />
 
-
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<br />
 
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<p><br /><h3>
 
-
<b>What is Spinach?</b></h3>
 
-
Spinach is a green fluorescent RNA sequence that can be expressed in cells (in this case, <i>E. coli </i>) that can be used to quantify RNA concentration in a cell. Spinach binds to an organic dye called DFHBI which doesn't fluoresce by itself but fluoresces very brightly when it is bound to Spinach. DFHBI is chemically derived from the chromophore in GFP but is altered to increase brightness when bound to RNA. Other fluorescent RNAs have been described but many are non-specific and have many unwanted functions like cytotoxicity. Manipulations to the sequence that Spinach is attached to allows for a variety of analyses functions. RNA can be arranged to bind to just about any small molecule in the same way that Spinach was developed (using a SELEX method) to track cellular metabolites. This allows for quantification of another important system in cells. However, in our system, Spinach is incorporated in the mRNA (between the promoter and the RBS) so mRNA is quantified. <a href="http://www.sciencemag.org/content/333/6042/642.full"> Click for more information on Spinach</a>. Spinach is the first published RNA sequence of its kind and more sequence/dye combinations are in development; as a result, in years to come, multiple genes (both RNA and protein) can be analyzed in great detail simultaneously.
 
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<br /><h3>
 
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<b>What is a FAP?</b></h3>
 
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A fluorogen activating protein is a small (26-35kD) protein that derives from the variable region in an antibody. FAPs are not fluorescent unless a fluorogen (also not normally fluorescent) is added, in which case the FAP changes the conformation of the fluorogen and the complex fluoresces brightly. FAPs are currently used to tag certain proteins like actin or tubulin in mammalian cells. FAPs are not primarily expressed in<i> E. coli</i> although we have expressed certain FAPs in <i>E. coli</i>. The two main dyes that the current series of FAPs bind to are malachite green and thiazole orange; our construct uses a variant that binds to malachite green. These dyes are normally cell impermeable but can be designed to penetrate cell membranes. As a result, they were originally used to tag surface proteins. FAPs are excellent reporters because they are small proteins that are soluble and have virtually no maturation time and are highly photostable unlike traditional variants of GFP. FAP technology is widely unexplored but shows promise for new fluorescent technology. FAPs have been used to track individual molecules to <a href="http://www.photonics.com/Article.aspx?AID=45024">5nm definition as opposed to the typical 200nm</a>. New dyes are being developed that can increase intensity and can allow researchers to improve on live cell imaging techniques. FAPs are genetically different so researchers can image multiple proteins at the same time in order to understand complex biological processes.
 
</p>
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<b>Why is this project important?</b><br />
 
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<p>
 
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The ability to monitor pathways in cells with fluorescence is a growing field that promises advances in drug development and drug manufacturing improvement. This technology allows scientists and engineers to understand how biological therapies are being made in culture. For example, certain drugs like monoclonal antibody therapies are produced in large bioreactors while cells produce the protein therapy. In order to optimize the protein production and do a quality control check, technicians can use this technology to ensure that cells are producing both the protein and the RNA. The FAP technology provides a method to analyze how the drugs are being secreted through the membrane into the bioreactor. This sort of technology allows manufacturers to tighten quality control on drugs with a very simple assay so that consistency between batches is optimized. </p>
 
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<p>
 
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This also allows a gateway into analyzing signaling pathways, particularly G-protein coupled receptor (GPCR) pathways. GPCR pathways are the target <a href="http://scienceblog.com/37030/carnegie-mellon-researchers-create-fluorescent-biosensor-to-aid-in-drug-development/">30%</a> of prescription drugs on the market and are involved in cell signaling. FAPs are currently used for this research and when this live cell imaging is supplemented with fluorescent RNA, researchers can understand how these pathways function and interact with transcription.</p>
 
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Our system is designed to be user-friendly for the typical synthetic biologist so that any project in need of an RNA reporter can use it.
 
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<a name="Primary_Objective:_A_Useful_BioBrick_for_Synthetic_Biologists"></a><h2> <span class="mw-headline"> Primary Objective: A Useful BioBrick for Synthetic Biologists </span></h2>
 
-
<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>Fluorescence Microscopy</div></div></div>
 
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<p>We believe the development of this unprecedented BioBrick will help synthetic biologists in a variety of applications,  <b>for a variety of purposes </b> such as the following:
 
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</p>
 
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<ol><li> Quantifying translational efficiency <i>in vivo </i>
 
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</li><li> Troubleshooting in expression strains
 
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</li><li> <i>in vivo</i> transcription rate analysis
 
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</li><li> Determining promoter strength <i> in vivo</i>
 
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</li><li> Distinguish between promoter strength and RBS strength
 
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</li><li> Determining <i>in vivo</i> mRNA and protein half-lives
 
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</li><li> Introducing a novel and promising protein reporter that has virtually no maturation rate and is limited only by the very quick absorption rate of the fluorogen into the cell
 
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</li><li> Introducing a functioning mRNA reporter and measurement BioBrick
 
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</li><li> Providing a novel method to characterize current and future BioBricks
 
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</li><li> Developing methods to analyze gene expression networks <i>in vivo</i> without disrupting behavior by fusing our construct to other proteins
 
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</li></ol>
 
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<p>Our proposed BioBrick is novel, and potentially very useful in practice.
 
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</p>
 
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<a name="Secondary_Objective:_Humanistic_Practice"></a><h2> <span class="mw-headline"> Secondary Objective: Humanistic Practice</span></h2>
 
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<p> <a href="https://2012.igem.org/Team:Carnegie_Mellon" class="external text" title="https://2012.igem.org/Team:Carnegie_Mellon" rel="nofollow">FAQ/Terminology</a> in engineering <i>Escherichia coli</i> to <b>monitor these variables via fluorescence</b>. Find out more about Carnegie Mellon: (<a href="http://www.cmu.edu" class="external text" title="http://www.cmu.edu" rel="nofollow">CMU Home Page</a>).
 
-
</p>
 
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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 to teach high school students about:
 
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<ol><li> Biological systems and synthetic biology
 
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</li><li> Gene expression and the central dogma of molecular biology
 
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</li><li> How our BioBrick can be used as a measurement system
 
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</li><li> How scientists tackle real-world problems using an interactive simulation that allows the use of our BioBrick and synthetic biological principles
 
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</li></ol>
 
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<a name="Further_Considerations"></a><h2> <span class="mw-headline"> Further Considerations </span></h2>
 
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<p>In the pursuit of our project, as well as the biological aspects, we:
 
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</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 modeling our BioBrick to students,
 
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</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 techniques for measuring translational efficiency and transcriptional strength,
 
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</li><li> Participated in human practices demonstration and modeled our biological system using a programmable and interactive, electrical analog.
 
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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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