Team:Virginia/Project

From 2012.igem.org

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<li class="toclevel-2 tocsection-4"><a href="#Bacteriophage_Basics"><span class="tocnumber">2.2</span> <span class="toctext">Bacteriophage Basics</span></a></li>
<li class="toclevel-2 tocsection-4"><a href="#Bacteriophage_Basics"><span class="tocnumber">2.2</span> <span class="toctext">Bacteriophage Basics</span></a></li>
<li class="toclevel-2 tocsection-5"><a href="#Bordetella_Phage"><span class="tocnumber">2.3</span> <span class="toctext">Bordetella Phage</span></a></li>
<li class="toclevel-2 tocsection-5"><a href="#Bordetella_Phage"><span class="tocnumber">2.3</span> <span class="toctext">Bordetella Phage</span></a></li>
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<li class="toclevel-2 tocsection-5"><a href="#T7_Phage"><span class="tocnumber">2.4</span> <span class="toctext">T7 Phage</span></a></li>
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Bacteriophage BPP-1 infects the genus <i>Bordetella</i>, which includes the human pathogen <i>B. pertussis</i> and its cousin <i>B. bronchiseptica</i>. It normally binds to the cell-surface receptor pertactin, which is only expressed during the Bvg+ phase of the <i>Bordetella</i> life cycle (during which the bacteria expresses virulence and colonization factors at high levels). However, BPP-1 also possesses a diversity-generating retroelement system which allows it to switch its tropism (Liu <i>et al.</i>, 2002, Liu <i>et al.</i>, 2004).
Bacteriophage BPP-1 infects the genus <i>Bordetella</i>, which includes the human pathogen <i>B. pertussis</i> and its cousin <i>B. bronchiseptica</i>. It normally binds to the cell-surface receptor pertactin, which is only expressed during the Bvg+ phase of the <i>Bordetella</i> life cycle (during which the bacteria expresses virulence and colonization factors at high levels). However, BPP-1 also possesses a diversity-generating retroelement system which allows it to switch its tropism (Liu <i>et al.</i>, 2002, Liu <i>et al.</i>, 2004).
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<h3><span class="editsection"></span> <span class="mw-headline" id="T7_Phage"> T7 Phage </span></h3>
<h2><span class="editsection"></span> <span class="mw-headline" id="References"> References </span></h2>
<h2><span class="editsection"></span> <span class="mw-headline" id="References"> References </span></h2>
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Revision as of 21:27, 3 October 2012





Contents

Project Overview

Whooping cough, the infectious respiratory disease caused by Bordetella pertussis, is diagnosed in tens of millions of people and results in almost 300,000 deaths globally each year. Low-income and unvaccinated individuals as well as infants are especially susceptible. Current diagnostic procedures are complicated, costly, and can take up to a week, by which time the disease may have progressed or spread. The enormous impact of this disease urgently motivates the development of a faster, cheaper, and more reliable diagnostic. Our epidemiology models suggest that earlier diagnosis could drastically reduce the incidence and impact of the disease. We propose an engineered bacteriophage diagnostic system for rapid clinical detection of pertussis.

As a proof of principle, we worked on engineering the T7 bacteriophage to express a version of the beta subunit of human chorionic gonadotropin (hCG) which we codon-optimized for expression in E. coli. Due to host-pathogen specificity, bacteriophages will only replicate and produce intracellular hCG if the target bacteria is present in the sample. The bacteriophage lyses the cell, releasing the hCG, which can then be detected using a pregnancy test. Pregnancy tests are commonly available in all clinics and can detect very low concentrations of hCG, which is an advantage over currently-available methods for detecting phage amplification. The bacteriophage system also requires minimal training to be used in lab and does not require any high-tech machinery. This will make the diagnostic readily usable in developing countries, where whooping cough is a particular concern. Besides pertussis, this method can be used to detect the presence of any bacteria for which there exists a bacteriophage using a common pregnancy test.


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Background

Limitations of Current Detection Methods

Bacteriophage Basics

Bacteriophage are viruses which specifically infect bacteria, hijacking the bacteria's own replication machinery to replicate themselves. Due to host-pathogen co-evolution, bacteriophage are often extremely specific to a particular genus, species, or even strain of bacteria. They may undergo the lytic cycle, in which the genome of the bacteriophage is used to produce new bacteriophage which are released from the bacterial cell following cell death and lysis, or the lysogenic cycle, in which the bacteriophage genome replicates along with the host genome but does not produce new viruses.

Bordetella Phage

Bacteriophage BPP-1 infects the genus Bordetella, which includes the human pathogen B. pertussis and its cousin B. bronchiseptica. It normally binds to the cell-surface receptor pertactin, which is only expressed during the Bvg+ phase of the Bordetella life cycle (during which the bacteria expresses virulence and colonization factors at high levels). However, BPP-1 also possesses a diversity-generating retroelement system which allows it to switch its tropism (Liu et al., 2002, Liu et al., 2004).

T7 Phage

References

1. Liu, M., Deora, R., Doulatov, S. R., Gingery, M., Eiserling, F. A., Preston, A., Maskell, D. J., et al. (2002). Reverse Transcriptase – Mediated Tropism Switching in Bordetella Bacteriophage. Science, 2091(295). doi:10.1126/science.1067467
2. Liu, M., Gingery, M., Doulatov, S. R., Liu, Y., Hodes, A., Baker, S., Davis, P., et al. (2004). Genomic and Genetic Analysis of Bordetella Bacteriophages Encoding Reverse Transcriptase-Mediated Tropism-Switching Cassettes. Journal of Bacteriology, 186(5), 1503–1517. doi:10.1128/JB.186.5.1503