Team:USC/Project

From 2011.igem.org

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<h1 style="font-family:Verdana;font-weight:500;">Overview</h1>
<h1 style="font-family:Verdana;font-weight:500;">Overview</h1>
Bacteria protect their genome and remove foreign DNA elements through the use of the clustered, regularly interspaced short palindromic repeat (CRIPSR) system. Recently discovered, this defense mechanism can protect microorganisms against bacteriaphages.  The CRISPR system comprises a transcribed locus of repeats and spacers that is activated and made functional via a family of CAS proteins. The CRISPR repeats are short DNA sequences that are almost identical in size and sequence. Between a pair of repeats is a spacer, which if complementary to a DNA element of the invading bacteriaphage, facilitates the destruction of that sequence from the bacterial cell.  As such, the CRISPR/CAS system is a primitive biological defense mechanism, similar in many ways to the process of adaptive immunity in eukaryotic immune systems.   
Bacteria protect their genome and remove foreign DNA elements through the use of the clustered, regularly interspaced short palindromic repeat (CRIPSR) system. Recently discovered, this defense mechanism can protect microorganisms against bacteriaphages.  The CRISPR system comprises a transcribed locus of repeats and spacers that is activated and made functional via a family of CAS proteins. The CRISPR repeats are short DNA sequences that are almost identical in size and sequence. Between a pair of repeats is a spacer, which if complementary to a DNA element of the invading bacteriaphage, facilitates the destruction of that sequence from the bacterial cell.  As such, the CRISPR/CAS system is a primitive biological defense mechanism, similar in many ways to the process of adaptive immunity in eukaryotic immune systems.   
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Plasmids, which are commonly used in molecular biology are also the source of horizontal gene transfer in the wild and facilitate the spread of antibiotic resistant genes among microorganisms.  These plasmids can contain unique DNA sequences that could be targeted by the CRISPR/CAS system.  We intend to exploit the CRISPR/CAS system to engineer a mechanism of plasmid curing and deactivation of antibiotic resistance genes in E. coli.
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Plasmids, which are commonly used in molecular biology are also the source of horizontal gene transfer in the wild and facilitate the spread of antibiotic resistant genes among microorganisms.  These plasmids can contain unique DNA sequences that could be targeted by the CRISPR/CAS system.  We exploit the CRISPR/CAS system to engineer a mechanism of plasmid curing and deactivation of antibiotic resistance genes in E. coli.
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To begin, our project will utilize a synthetic version of CRISPR generated by inserting an engineered spacer that matches the DNA sequence coded within an E. coli expression plasmid. We will first test the synthetic CRISPR array against E. coli harboring a tetR regulated tetO::GFP system. We anticipate that the CRISPER tetR targeted spacer will destroy the tetR plasmid, relieving the inhibition of the tetO locus and facilitating the expression of GFP. In the near future, we hope to use the synthetic CRISPR system as a biological tool, combining it with other BioBricks and parts for use in many applications that will impact health and medicine, biotechnology, molecular biology and genetics.
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We synthesized a version of CRISPR encoding a spacer that matches the GFP DNA sequence. We tested the synthetic CRISPR array against E.coli harboring a tetO::GFP plasmid that confers ampicillin resistance. Activation of CRISPER-GFP destroys the GFP-containing plasmid restoring the bacterial host's sensitivity to ampicillin. We will use the synthetic CRISPR system as a biological tool, combining it with other BioBricks for use in applications that will impact health and medicine, biotechnology, molecular biology, and genetics.
<h1 style="font-family:Verdana;font-weight:500;">Details</h1>
<h1 style="font-family:Verdana;font-weight:500;">Details</h1>
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The design of our project takes advantage of E. coli as a platform.  
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Studies have shown that ''Escherichia coli'' bacteria have a system that provides immunity against viral infections and plasmid conjugation in the natural world. This recently discovered system, also known as the CRISPR/Cas system, is made of eight cas (CRISPR-associated) genes and an array of 
[[File:CRISPRworks.png|600px|thumb|center|alt=Alt text|Possible mechanism of CRISPR/cas system against viral DNA]]
[[File:CRISPRworks.png|600px|thumb|center|alt=Alt text|Possible mechanism of CRISPR/cas system against viral DNA]]
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<h1 style="font-family:Verdana;font-weight:500;">Results</h1>
<h1 style="font-family:Verdana;font-weight:500;">Results</h1>
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We tested the synthetic version of
 
<h1 style="font-family:Verdana;font-weight:500;">CRISPR/cas System in K12 E. coli</h1>
<h1 style="font-family:Verdana;font-weight:500;">CRISPR/cas System in K12 E. coli</h1>

Revision as of 01:00, 29 September 2011

USC Banner.jpg


Contents

Project

Overview

Bacteria protect their genome and remove foreign DNA elements through the use of the clustered, regularly interspaced short palindromic repeat (CRIPSR) system. Recently discovered, this defense mechanism can protect microorganisms against bacteriaphages. The CRISPR system comprises a transcribed locus of repeats and spacers that is activated and made functional via a family of CAS proteins. The CRISPR repeats are short DNA sequences that are almost identical in size and sequence. Between a pair of repeats is a spacer, which if complementary to a DNA element of the invading bacteriaphage, facilitates the destruction of that sequence from the bacterial cell. As such, the CRISPR/CAS system is a primitive biological defense mechanism, similar in many ways to the process of adaptive immunity in eukaryotic immune systems. Plasmids, which are commonly used in molecular biology are also the source of horizontal gene transfer in the wild and facilitate the spread of antibiotic resistant genes among microorganisms. These plasmids can contain unique DNA sequences that could be targeted by the CRISPR/CAS system. We exploit the CRISPR/CAS system to engineer a mechanism of plasmid curing and deactivation of antibiotic resistance genes in E. coli.

We synthesized a version of CRISPR encoding a spacer that matches the GFP DNA sequence. We tested the synthetic CRISPR array against E.coli harboring a tetO::GFP plasmid that confers ampicillin resistance. Activation of CRISPER-GFP destroys the GFP-containing plasmid restoring the bacterial host's sensitivity to ampicillin. We will use the synthetic CRISPR system as a biological tool, combining it with other BioBricks for use in applications that will impact health and medicine, biotechnology, molecular biology, and genetics.

Details

Studies have shown that Escherichia coli bacteria have a system that provides immunity against viral infections and plasmid conjugation in the natural world. This recently discovered system, also known as the CRISPR/Cas system, is made of eight cas (CRISPR-associated) genes and an array of

Alt text
Possible mechanism of CRISPR/cas system against viral DNA
CRISPRlocus.png
CRISPRschematic.png
N-CRISPR.png
CRISPRsetup.png

Results


CRISPR/cas System in K12 E. coli

HT115grow1.png
HT115g1.jpeg
HT115g2.png
HT115grow2.png
HT115grow3.png

CRISPR/cas System in B E. coli

BL21grow1.png
B1B2B3.jpeg
Gfpb1b2b3.jpeg
BL21grow2.png
D1D2D3.jpeg
Gfpd1d2d3.jpeg
BL21grow3.png
E1E2E3.jpeg
Gfpe1e2e3.jpeg
BL21grow4.png
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