Team:USP-UNESP-Brazil/Plasmid Plug n Play/Introduction

From 2012.igem.org

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This project goal is to build a machine for producing proteins (called Plug&Play). This machine will be a tool for rapid protein expression, which would decrease the time used in screening candidate genes that could encode proteins with functions of biological interest. It only uses parts found in the Registry of Standard Biological Parts created by the iGEM competition, allowing its free use by the synthetic biology community.
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This project goal is to build a machine for producing proteins, called Plug&Play. This machine will be a tool for an easy and rapid protein expression, which aims to decrease the time used in screening candidate genes that encode proteins of biological interest. It only uses parts from the Registry of Standard Biological Parts, allowing its free use by the synthetic biology community.
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As a proof of concept we proposed to build a single plasmid that allows any protein expression in ''E. coli'' using two steps: PCR (Polymerase Chain Reaction) and transformation. The system is based on the Cre recombinase protein that catalyzes DNA recombination between specific sites.
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As a proof of concept we proposed to build a single plasmid that allows the expression of any protein in ''E. coli'' using two steps: PCR (Polymerase Chain Reaction) and bacteria transformation. The system is based on the Cre recombinase protein that catalyzes DNA recombination between specific recognition sites.
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The Plug&Play system has two parts;
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The Plug&Play system consist of two parts;
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i)    primers for amplifying the desired ORF (open reading frame) flanked by the recombination sequences loxP and lox66, which are recognized by the Cre recombinase.
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i)    primers to amplify the desired ORF (open reading frame) - the primers sequences must be flanked by the recombination sequences loxP and lox66, which will be recognized by the Cre recombinase.
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ii)    a receptor plasmid that uses the Cre recombination mechanism to insert the sequence amplified by PCR in a specific place (lox71 site), which already posses all the necessary machinery to express the protein.
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ii)    a receptor plasmid - the Cre recombination mechanism will strategically insert the PCR-amplified DNA at the lox71 site, and readily express the protein once the receptor plasmid already posses all the necessary protein expression machinery.
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We designed three plasmid versions to be tested; one using a low copy number plasmid (pSB4A5), one using a high copy number plasmid (pSB1C3) and one using a commercial high copy number plasmid (pET15b). These versions allow us to compare which could be the best system for developing this technology.  
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We intended to test three different plasmids: a low copy number plasmid (pSB4A5), a high copy number plasmid (pSB1C3) and a commercial high copy number plasmid (pET15b). The comparison of these three plasmids will identify the best system for the developing of this technology.  
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The Cre expression levels are critical for the system performance, for this reason we created an A (Cre-Lox71) and B (Lox71-Cre) versions of the three plasmid constructions. We wanted to test if the target gene insertion upstream or downstream the Cre gene, into the receptor plasmid, could affect the expression efficiency.
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Since the Cre expression levels are critical for the system performance, we created two different version for each of the three plasmids, A (Cre-Lox71) and B (Lox71-Cre). We want to test if inserting the target gene either upstream or downstream the Cre gene will affect the expression efficiency.

Revision as of 20:43, 24 September 2012