Team:Arizona State/Chimeric Reporter

From 2012.igem.org

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<h1>DNA-Protein Chimera Biosensor</h1>
<h1>DNA-Protein Chimera Biosensor</h1>
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<h2>Overview</h2>
<h2>Overview</h2>
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There are various biosensors on the market but the state of the art technology is based upon Polymerase Chain Reaction and nanotechnology, which involves gold plated probes and requires specialized skills to use. Despite the extreme accuracy of the device, the affordability, and longer diagnostic time has made the technology scarce in the field. In order to make biosensing technology more accessible to those with few resources and the greatest need, the team worked on generating a cost effective, highly accurate and user-friendly organic biosensor. The components of the sensor will be produced in non-pathogenic E. coli. The sensor is made up of a protein head and DNA tail. The protein head is an enzyme that turns a colorless substrate (X-gal) blue. The enzyme is split in half, so that when the sensor is dissolved in water it cannot produce blue color. When pathogenic target DNA is present, two DNA sensor tails bind the target, the split enzyme assembles, and blue color is produced. Color provides a user-friendly output that is familiar to non-skilled users.  
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There are various biosensors on the market but the state of the art technology is based upon Polymerase Chain Reaction and nanotechnology, which involves gold plated probes and requires specialized skills to use. Despite the extreme accuracy of the device, the affordability, and longer diagnostic time has made the technology scarce in the field. In order to make biosensing technology more accessible to those with few resources and the greatest need, the team worked on generating a cost effective, highly accurate and user-friendly organic biosensor. The components of the sensor will be produced in non-pathogenic <i>E. coli</i>.  
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<h2>Streptavidin</h2>
<h2>Streptavidin</h2>
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Purified and extracted from the bacteria Streptomyces avidinii, Streptavidin posses a high binding affinity for biotin, with a dissociation constant of 10^-14–10^–16 M ( Laitinen et al.). With such high dissociation constant, the bonding of streptavidin to biotin is considered as one of the strongest non covalent bonding in nature. Due to the high binding affinity to biotin streptavidin serves as one of the major component of this project.
 
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As a proof of concept for our design, our team designed and assembled fusions of streptavidin (strep) and the split beta-galactosidase (bgal) fragments. Streptavidin has a high binding affinity to biotin, so it will our fusion proteins to easily bind onto the ends of biotinylated DNA fragments.
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Purified and extracted from the bacteria Streptomyces avidinii, streptavidin possesses a high binding affinity for biotin, with a dissociation constant of 10^-14–10^–16 M ( Laitinen et al.). With such a low dissociation constant, the bonding of streptavidin to biotin is considered to be one of the strongest non-covalent bonds in nature. This high binding affinity to biotin will ensure the integrity of the biosensor.
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The addition of a poly-histidine tag (His-tag) makes it possible to generate the fusion proteins in E. Coli, then isolate and purify them using a nickle binding column. Mixtures of His-purified strep-tagged bgal fragments and single stranded biotinylated DNA will generate DNA-Protein Chimera 'probes' as the streptavidin binds to the biotinylated end of the DNA.  
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As a proof of concept for our design, our team designed and assembled fusions of streptavidin (strep) and the split beta-galactosidase (bgal) fragments. Because streptavidin binds to biotin with high affinity, it will allow our fusion proteins to easily bind onto the ends of biotinylated DNA fragments.
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</p>
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<p>
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The addition of a poly-histidine tag (His-tag) makes it possible to generate the fusion proteins in E. Coli, then isolate and purify them using a nickel binding column. Mixtures of His-purified strep-tagged bgal fragments and single stranded biotinylated DNA will generate DNA-Protein Chimera 'probes' as the streptavidin binds to the biotinylated end of the DNA.  
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</p>
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<p>
By creating probes using complementary strands of DNA of varying lengths, we can confirm that DNA-Protein Chimeric probes generated in E. Coli will create a colorimetric response when kept in close proximity. This will allow us to characterize the behavior of split bgal fusion probes as a function of the distance that they are separated - which can be controlled by altering the length of the ssDNA, or by creating two probes that bind to the same template ssDNA at different sites separated by a variable length.
By creating probes using complementary strands of DNA of varying lengths, we can confirm that DNA-Protein Chimeric probes generated in E. Coli will create a colorimetric response when kept in close proximity. This will allow us to characterize the behavior of split bgal fusion probes as a function of the distance that they are separated - which can be controlled by altering the length of the ssDNA, or by creating two probes that bind to the same template ssDNA at different sites separated by a variable length.
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<h2>Topoisomerase</h2>
<h2>Topoisomerase</h2>
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<img src="https://static.igem.org/mediawiki/2012/8/8f/TopoDiagram.png" width="800" height="500">
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<img src="https://static.igem.org/mediawiki/2012/8/8f/TopoDiagram.png" width="900" />
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The wild type form of topoisomerase binds to the DNA sequence (YCCTT) in E. Coli. It regulates the winding of the DNA by making a nick after the second T. This allows for the rotation of the strands to relieve torsional stress. Afterwards, the DNA strands are religated. In 2006, Bushman et al. have shown that the smallpox topoisomerase double cysteine mutant D168A mutates the tyrosine responsible for covalent bonding to the 5’ phosphate at the DNA nicking. This mutant form prevents religation, and thus causes the majority of the DNA to stay in the covalently bonded complex.
The wild type form of topoisomerase binds to the DNA sequence (YCCTT) in E. Coli. It regulates the winding of the DNA by making a nick after the second T. This allows for the rotation of the strands to relieve torsional stress. Afterwards, the DNA strands are religated. In 2006, Bushman et al. have shown that the smallpox topoisomerase double cysteine mutant D168A mutates the tyrosine responsible for covalent bonding to the 5’ phosphate at the DNA nicking. This mutant form prevents religation, and thus causes the majority of the DNA to stay in the covalently bonded complex.
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<h2>Design Scheme</h2>
<h2>Design Scheme</h2>
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In our design, we plan to use topoisomerase to nick a specific covalently bonded sequence and peel off a section of single stranded DNA. We have designed a template plasmid that includes tandem YCCTT recognition sites with template strand in between, and is complementary to a section of coding sequence of GFP. We plan to use a KEIO strain with one copy of this coding sequence in the E. Coli genome.
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In our design, we plan to use topoisomerase to nick a specific covalently bonded sequence and peel off a section of single stranded DNA. We have designed a template plasmid that includes tandem YCCTT recognition sites with template strand in between, and is complementary to a section of coding sequence of GFP. By inducing topoisomerase/split bgal fusion protein expression, we will be able to generate Chimeric probes <i>in vivo</i> that can be easily His-tag purified and tested. We plan to use a KEIO strain with one copy of this coding sequence in the <i>E. Coli</i> genome in order to test the function of our chimeric probes on cell lysates and mock water samples.
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<h2>Reporter System</h2>
<h2>Reporter System</h2>
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<p>
<p>
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Basilion et al. from Case Western in 2010 have shown that they were able to make a split beta-galactosidase complementation assay with relatively reliable assay results In the assay, alpha-4/omega, which has a higher specificity, is the most successful split beta galactosidase assay. It is thus used to eliminate false positive. Additionally, we are adapting alpha and 1-omega, which is less specific but has a higher signal, for the same protocol to eliminate false negative.
+
Basilion et al. from Case Western in 2010 have shown that they were able to make a split beta-galactosidase complementation assay with relatively reliable assay results. In the assay, alpha-4/omega, which has a higher specificity, is the most successful split beta galactosidase assay. It is thus used to eliminate false positive. Additionally, we are adapting alpha and 1-omega, which is less specific but has a higher signal, for the same protocol to eliminate false negative.
</p>
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<p>
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Additionally, Basilion et al. also demonstrated success in creating fusion proteins with a split-beta galactosidase fragment and antibody specific to their target. Modifying this, we plan to make a fusion of our mutant topoisomerase and our split-beta galactosidase fragments. This effectively creates a probe that when assembled contains topoisomerase bound both to a single stranded DNA hybridization probe and a split-beta galactosidase fragment. By incubating the two probes that recognize adjacent DNA sequences, we can test for the presence of DNA sequences in a bacterial genome.
+
Basilion et al. also demonstrated success in creating fusion proteins with a split-beta galactosidase fragment and antibody specific to their target. Modifying this, we plan to make a fusion of our mutant topoisomerase and our split-beta galactosidase fragments. This effectively creates a probe that when assembled contains topoisomerase bound both to a single stranded DNA hybridization probe and a split-beta galactosidase fragment. By incubating the two probes that recognize adjacent DNA sequences, we can test for the presence of DNA sequences in a bacterial genome.
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Latest revision as of 00:30, 27 October 2012


DNA-Protein Chimera Biosensor

Overview

There are various biosensors on the market but the state of the art technology is based upon Polymerase Chain Reaction and nanotechnology, which involves gold plated probes and requires specialized skills to use. Despite the extreme accuracy of the device, the affordability, and longer diagnostic time has made the technology scarce in the field. In order to make biosensing technology more accessible to those with few resources and the greatest need, the team worked on generating a cost effective, highly accurate and user-friendly organic biosensor. The components of the sensor will be produced in non-pathogenic E. coli.


Streptavidin

Purified and extracted from the bacteria Streptomyces avidinii, streptavidin possesses a high binding affinity for biotin, with a dissociation constant of 10^-14–10^–16 M ( Laitinen et al.). With such a low dissociation constant, the bonding of streptavidin to biotin is considered to be one of the strongest non-covalent bonds in nature. This high binding affinity to biotin will ensure the integrity of the biosensor.

As a proof of concept for our design, our team designed and assembled fusions of streptavidin (strep) and the split beta-galactosidase (bgal) fragments. Because streptavidin binds to biotin with high affinity, it will allow our fusion proteins to easily bind onto the ends of biotinylated DNA fragments.

The addition of a poly-histidine tag (His-tag) makes it possible to generate the fusion proteins in E. Coli, then isolate and purify them using a nickel binding column. Mixtures of His-purified strep-tagged bgal fragments and single stranded biotinylated DNA will generate DNA-Protein Chimera 'probes' as the streptavidin binds to the biotinylated end of the DNA.

By creating probes using complementary strands of DNA of varying lengths, we can confirm that DNA-Protein Chimeric probes generated in E. Coli will create a colorimetric response when kept in close proximity. This will allow us to characterize the behavior of split bgal fusion probes as a function of the distance that they are separated - which can be controlled by altering the length of the ssDNA, or by creating two probes that bind to the same template ssDNA at different sites separated by a variable length.


Topoisomerase

The wild type form of topoisomerase binds to the DNA sequence (YCCTT) in E. Coli. It regulates the winding of the DNA by making a nick after the second T. This allows for the rotation of the strands to relieve torsional stress. Afterwards, the DNA strands are religated. In 2006, Bushman et al. have shown that the smallpox topoisomerase double cysteine mutant D168A mutates the tyrosine responsible for covalent bonding to the 5’ phosphate at the DNA nicking. This mutant form prevents religation, and thus causes the majority of the DNA to stay in the covalently bonded complex.


Design Scheme

In our design, we plan to use topoisomerase to nick a specific covalently bonded sequence and peel off a section of single stranded DNA. We have designed a template plasmid that includes tandem YCCTT recognition sites with template strand in between, and is complementary to a section of coding sequence of GFP. By inducing topoisomerase/split bgal fusion protein expression, we will be able to generate Chimeric probes in vivo that can be easily His-tag purified and tested. We plan to use a KEIO strain with one copy of this coding sequence in the E. Coli genome in order to test the function of our chimeric probes on cell lysates and mock water samples.


Reporter System

Basilion et al. from Case Western in 2010 have shown that they were able to make a split beta-galactosidase complementation assay with relatively reliable assay results. In the assay, alpha-4/omega, which has a higher specificity, is the most successful split beta galactosidase assay. It is thus used to eliminate false positive. Additionally, we are adapting alpha and 1-omega, which is less specific but has a higher signal, for the same protocol to eliminate false negative.

Basilion et al. also demonstrated success in creating fusion proteins with a split-beta galactosidase fragment and antibody specific to their target. Modifying this, we plan to make a fusion of our mutant topoisomerase and our split-beta galactosidase fragments. This effectively creates a probe that when assembled contains topoisomerase bound both to a single stranded DNA hybridization probe and a split-beta galactosidase fragment. By incubating the two probes that recognize adjacent DNA sequences, we can test for the presence of DNA sequences in a bacterial genome.

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