Difference between revisions of "Team:Washington/Auxin"

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                 <h2>
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                     <div id="Results">What are the goals of the project?</div>
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                     <div id="Results">Test Strip Design</div>
 
                 </h2>
 
                 </h2>
  
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                 <p>1. Develop a paper microfluidic device that houses yeast; it will provide adequate nutrients for cell growth, but will also be freeze-dried for long-term storage.
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                 <p>The design for the yeast biosensor needs to create an ideal environment for the culture.</p>
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                <p>2. Create a detection system for the plant hormone auxin in which yeast produce a color in response to an auxin input; test it in cells growing on normal media.
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                 <p>The base of the test strip, chromatography paper, The PDMS is ideal for a test strip because it is manufactured rapidly at a low cost. The PDMS window allows small molecule-gasses to permeate but not foreign contaminants. The one-way valve would most likely have a connection to a small pipette that could deposit medium evenly across the yeast cells. The chromatography paper spreads the test solution evenly so that different sections of the yeast media have the same concentration of test solution. In this way, the indigo color will be even and predictable in the yeast section. A section of the strip will have yeast that constitutively expresses indigo as a control to ensure that the rehydration is functional.</p>
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                <p>3. Clone each part from this system into standard plasmids for submission to BioBrick registry.
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                <p>4. Show that when grown on paper, yeast can reliably detect auxin.
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                 <p>5. Use aptamers (short DNA sequences) to detect for okadaic acid, a shellfish toxin that causes Diarrhetic Shellfish Poisoning.
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                <p>6. Show that the aptamer system for detection can be implemented on our paper platform and reliably detect for okadaic acid.
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                <p>7. Improve safety of shellfish consumers in the NW and the world!
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                <h2> Lab on a Strip: Developing a Novel Platform for Yeast Biosensors </h2>
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                <h2>Overview </h2>
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                <p>Biosensors for detecting small molecules have many applications in medicine, food, and the environment. Our project aims to combine the emerging fields of synthetic biology and paper diagnostics to create an affordable and accessible platform for a new class of biological sensors that could detect a wide variety of molecules. We first developed a paper microfluidic device housing Saccharomyces cerevisiae, which was then modified to accommodate two different biological detection systems. In one system, the Auxin/IAA-Degron pathway is used in conjunction with beta-galactosidase to produce a visible signal in response to the plant hormone auxin. In the other system, aptazymes, a combination of RNA aptamers and ribozymes, are used to bind theophylline and allow fluorescent protein to be produced. Both pathways serve as models for future real-world applications of our device, including the detection of marine biotoxins in the Pacific Northwest.  </p>
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                <p>For commercial shellfish farmers and recreational hunters alike, marine biotoxins pose a significant threat to health and welfare. With this project, we aim to create an inexpensive and easy-to-use test kit for the detection of the shellfish toxin okadaic acid using engineered yeast strains and DNA aptamers on a paper device. We also hope that this project paves the way for a new class of biosensors capable of detecting a wide range of small molecules. </p>
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                <h2>
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                    <div id="Parts">What is the context of this research?</div>
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                </h2>
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                <p>Marine toxins are an increasing problem in Washington State waters. Produced in high concentrations by microorganisms during algae blooms, they are ingested by filter-feeding shellfish, causing illness and death in human consumers. Biotoxins are also difficult to detect; contrary to popular belief, algal blooms are not always the striking crimson of “red tides.” Thus, blooms may not be discovered until after a poisoned shellfish is found. The Washington State Department of Health and commercial shellfish farmers conduct periodic surveys of local beaches to catch contaminations early, but these methods are costly, time-consuming, and not always effective. This can especially pose a dilemma for individual shellfish hunters, who do not have the resources to screen their shellfish for toxins. </p>
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                <h2>What is the significance of this project? </h2>
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                <p>Our project aims to combine the emerging fields of synthetic biology and paper diagnostics to create an affordable, accessible, and accurate diagnostic test kit that would allow farmers and the public to test shellfish for common biotoxins. This “lab on a strip” will be a critical step forward in marine toxin detection, as it will cut nearly 20 hours off the time needed to obtain results, allowing farmers to screen at a lower cost and empowering individual hunters to confirm the safety of their shellfish. This is also the first project to attempt to grow yeast on a paper device, and if successful, could open the door to a wide range of similar biosensors. Such sensors would have applications in medicine, food, and the environment worldwide. </p>
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                <h2>What are the goals of the project?</h2>
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                <h2>Lateral Flow Test Strip Background</h2>
 +
                <p>In a typical lateral flow assay, there are enzymes in the test strip that are in a dried salt and sugar mix. </p>
 +
                <p>While the sample fluid dissolves the salt-sugar matrix, it dissolves the particles and the sample and enzymes and salt and sugar mix. The analyte binds to the particles while traveling to the third capillary bed. This material has one or more areas (often stripes) where a third molecule has been placed by the manufacturer. By the time the sample mix reaches these strips, the analyte has been bound to the enzyme and the third 'capture' molecule binds the complex, and changes color. The color increases as enzyme-analyte-third molecules accumulate. </p>
 +
              <h2>Saccharomyces Cerivisiae Background</h2>
 +
                <p> The type of cell that was engineered is Saccharomyces Cerivisiae, commonly known as baker’s yeast.  This cell has an activity > 0 for pH from 2.1 to 7 (Arroyo et al. 2009).  The activity is above 0 and increasing from 12°C to 36°C (Arroyo et al. 2009).  The wild type Saccharomyces Cerivisiae is not known to be mutagenic.  Another crucial characterization for test strip media is longevity.  S. Cerivisiae can live for approximately 20 to 120 hours (Minois et al. 2004).  In a dehydrated dormant state, however, the yeast can survive for years (Fabrizio & Longo 2003).  The genome of this model organism has been sequenced, it is easy to obtain in the lab, and the genomic structure is easy to modify.</p>
  
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Revision as of 06:07, 16 September 2015



Overview

Prior CRISPR transcriptional factors

CRISPR transcriptional factors are a breakthrough because they enable control of the expression of a particular gene – based on the gRNA. In these systems, the gRNA is attached to a CAS (CRISPR associated) protein, often CAS9. These proteins are degraded so that they do not cleave the dsDNA. The CAS9 protein is attached to either a repressor or an enhancer, which modulates the expression of the gene. Ubiquitination enables a system that can change only once. CRISPR transcriptional factors were first developed by Perez-Pinera et al. in 2013.

Auxin Background

Auxin-IAA is a plant hormone that signals the development of leaves. This molecule serves as a model molecule for detection by the CRISPR transcriptional factors because it can pass through the membrane and because it has well-characterized corresponding F-box (AFB2) and degron (deg1). IAA is used in almost all plants, and is created by the from the amino acid tryptophan, and the synthesis is well characterized.

Design:

The pathway relies on CRISPR transcription factors to produce an indigo response to the plant hormone auxin-iaa (indole-3 acetic acid).

The Cas9 with a deactivated nuclease, or dCas9, binds to a gRNA strand that is complementary to the region of S. Cerivisiae that is near the inserted lacZ gene. Mxi1 is a repressor protein. In the presence of auxin, the degron protein recruits an F-box and a ubiquitin ligase (2). This E3 ubiquitin ligase tags the protein complex for degredation.

The β-galactosidase enzyme protein (3) catalyzes the separation of X-gal into galactose and 5-bromo-4-chloro-3-hydroxyindole. The 5-bromo-4-chloro-3-hydroxyindole then dimerizes and is oxidized to form the visible blue color.

The CRISPR transcriptional factor is an optimal method for sensing molecules because the components can be optimized and substituted. In this case, the AFB2 protein serves as the F-box and the Deg1 protein serves as the degron. Also, this system is applicable to other genes and organisms with dsDNA.

Test Strip Design

The design for the yeast biosensor needs to create an ideal environment for the culture.

The base of the test strip, chromatography paper, The PDMS is ideal for a test strip because it is manufactured rapidly at a low cost. The PDMS window allows small molecule-gasses to permeate but not foreign contaminants. The one-way valve would most likely have a connection to a small pipette that could deposit medium evenly across the yeast cells. The chromatography paper spreads the test solution evenly so that different sections of the yeast media have the same concentration of test solution. In this way, the indigo color will be even and predictable in the yeast section. A section of the strip will have yeast that constitutively expresses indigo as a control to ensure that the rehydration is functional.

Lateral Flow Test Strip Background

In a typical lateral flow assay, there are enzymes in the test strip that are in a dried salt and sugar mix.

While the sample fluid dissolves the salt-sugar matrix, it dissolves the particles and the sample and enzymes and salt and sugar mix. The analyte binds to the particles while traveling to the third capillary bed. This material has one or more areas (often stripes) where a third molecule has been placed by the manufacturer. By the time the sample mix reaches these strips, the analyte has been bound to the enzyme and the third 'capture' molecule binds the complex, and changes color. The color increases as enzyme-analyte-third molecules accumulate.

Saccharomyces Cerivisiae Background

The type of cell that was engineered is Saccharomyces Cerivisiae, commonly known as baker’s yeast. This cell has an activity > 0 for pH from 2.1 to 7 (Arroyo et al. 2009). The activity is above 0 and increasing from 12°C to 36°C (Arroyo et al. 2009). The wild type Saccharomyces Cerivisiae is not known to be mutagenic. Another crucial characterization for test strip media is longevity. S. Cerivisiae can live for approximately 20 to 120 hours (Minois et al. 2004). In a dehydrated dormant state, however, the yeast can survive for years (Fabrizio & Longo 2003). The genome of this model organism has been sequenced, it is easy to obtain in the lab, and the genomic structure is easy to modify.