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

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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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<h1 id="The Plug&Play Machine">The Plug&Play Machine</h1>
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As a proof of concept we proposed to build a single plasmid that allows any protein in ''E. coli'' expression in 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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The goal of this project is to build a prototype machine to produce/screen biological parts in high-throughput manner. This is the first step for characterizing/producing new biological standard parts, which is a huge necessity in the synthetic biology field. To help in this task we developed a prototype plasmid for a machine called Plug&Play, it express any protein helped by the Cre-Recombinase system. 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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The Plug&Play system has two parts;
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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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i)    primers for amplifying the desired ORF (open reading frame) flanked by the recombination sequences loxP and lox66, recognized by the Cre recombinase.
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The Plug&Play system consist of two parts;
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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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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 - 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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{{:Team:USP-UNESP-Brazil/Templates/RImage | image=ORFinsertion.png | caption=ORF insertion in Plug&Play plasmid | size=600px}}
 
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{{:Team:USP-UNESP-Brazil/Templates/RImage | image=Pplay_fig.jpeg | caption=ORF circularization and insertion in the Plug&Play plasmid | size=600px}}
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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 (pGEM). These versions allow us to compare which could be the best system for developing 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 efficiency of the expression.
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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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Since the Cre expression levels are critical for the system performance, we created two different versions for each of the three plasmids, A (lox71-Cre) and B (Cre-lox71). We want to test if inserting the target gene either upstream or downstream the Cre gene will affect the expression efficiency.
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Latest revision as of 22:29, 25 September 2012