Team:UCLA/Project

From 2013.igem.org

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<h4>How does it work?</h4>
<h4>How does it work?</h4>
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-First generate a library of DNA using pcr or some other method.
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<ul>
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-Next DNA library is transcribed in vivo to an RNA library.
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<li>First generate a library of DNA using pcr or some other method.</li>
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-With the help of a DNA splint, this RNA strand is ligated to a single strained DNA that    contains a puromycin protein at the 3 ' end
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<li>Next DNA library is transcribed in vivo to an RNA library.</li>
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<li>With the help of a DNA splint, this RNA strand is ligated to a single strained DNA that    contains a puromycin protein at the 3 ' end</li>
-This RNA-puromycin fusion is then translated in vivo.
-This RNA-puromycin fusion is then translated in vivo.
-The Puromycin mimics a tRNA and is incorporated into the A site of the ribosome. However the puromycin has a non hydrolizable amide bond, therefore terminating translation and releasing the polypeptide, which is still bound to the RNA template through the puromycin.
-The Puromycin mimics a tRNA and is incorporated into the A site of the ribosome. However the puromycin has a non hydrolizable amide bond, therefore terminating translation and releasing the polypeptide, which is still bound to the RNA template through the puromycin.
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-Once you have your protein, it can be easily identified because it is still bound to the DNA that coded for it.
-Once you have your protein, it can be easily identified because it is still bound to the DNA that coded for it.
-Simple pcr of the DNA followed by sequencing will give you the identity of your protein, (ie the sequence that codes for it.)
-Simple pcr of the DNA followed by sequencing will give you the identity of your protein, (ie the sequence that codes for it.)
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</ul>

Revision as of 19:02, 24 September 2013

Contents

Overall project

In-Vitro Immune System

Diversity is an intrinsic characteristic of nature, and it can be harnessed to serve as a useful tool in synthetic biology.

In particular, this diversity is exemplified in a species of bacteriophage known as the Bordetella BPP-1 phage. The Bordetella bacteria, a genus of small Gram-negative bacteria, frequently changes its surface topography over the course of its life cycle. To successfully infect its constantly-changing host, the BPP-1 phage uses diversity as its weapon of choice. The tip of each virus tail-fiber is a protein called the major tropism-determinant protein (mtd), which binds to the hosts’ surface and allows the phage to infect it.

Though the general structure of the mtd protein is constant and is very stable, its active ends are extremely variable. Each phage produces a slightly different structural variant of the protein, with the hope that at least one phage can successfully bind to the host and infect it- akin to the way our immune system produces numerous variants of antibodies so that at least one can bind to the antigen of interest.

The mtd gene, extracted from the BPP-1 phage genome, has the capability of generating a diverse library of stable proteins. Our team hopes to develop a fully in-vitro system to express and select for mtd variants of interest.

We plan on first generating a diverse DNA library of the Mtd gene, then using mRNA-display to detect and screen for the variants of the protein that can bind to a selected target. In our case, the target will be the surface of E. Coli bacterial cells.

Background Information

The Natural Host

Bordetella is the natural host of the BPP-1 bacteriophage. It expresses a protein on its surface, pertactin, that BPP-1 binds to in the first step of the infection process. However, Bordetella does not always express pertactin. It cycles between two phases: Bvg+ and Bvg-. Pertactin is only expressed in the Bvg+ phase. In the Bvg- phase, pertactin expression is inhibited. Infection by BPP-1 during this phase is much less common, but it still occurs. This indicates that the BPP-1 virus has a mechanism for changing its host specificity, and thus, the binding properties of the proteins on its tail fibers.

The Virus

The BPP-1 virus is a bacteriophage belonging to the Podoviridae family. It has an icosahedral head with T=7 symmetry, and a short, noncontractile tail with six tail “spikes” attached to tail fibers. At the end of these tail fibers are Major Tropism Determinant (mtd) proteins, which bind to the pertactin proteins expressed on the surface of Bordetella. The mtd protein does not display high affinity for pertactin, but the multiple mtd proteins possessed by each phage particle coupled with the flexibility of the tail fibers allow BPP-1 as a whole to have high avidity for its host. Following infection, the virus is lytic, and destroys the bacterial host to release more copies of itself.

mRNA Display

Historical Background

Richard Roberts and Jack Szostak from Harvard developed this technique in 1997. They were able to generate and identify a myc epitope starting from a random mrna library using this technique.

What is it?

mRNA display is a technique used to generate and select for proteins that bind specific targets. MRNA display allows for the identification of these selected proteins because they are covalently attached to the DNA that codes for them.

How does it work?




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