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

From 2012.igem.org

The objective of this project 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 a free use by the synthetic biology community.

It is proposed, as a proof of concept, the construction of a single plasmid that allows the expression of any protein in E. coli in two steps: PCR (Polymerase Chain Reaction) and transformation. The system is based on the Cre recombinase protein that catalyzes the recombination of DNA between specific sites in a DNA molecule.

The Plug & Play system has two parts; i) primers for amplifying the sequence of the desired ORF (open reading frame) containing the recombination sequence recognized by the Cre enzyme

ii) a receptor plasmid that uses the Cre recombination mechanism to insert the sequence amplified by PCR in a specific place, which already posses all the necessary machinery to express the protein.

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We design 3 versions of the plasmid 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 (pGEM). These versions allow us to compare which could be the best system for developing this technology.

The levels of Cre expressed are critical for the system performance, for this reason we created an A (Cre-Lox71) and B (Lox-Cre) constructions from each of the three plasmid versions. We wanted to test if the insertion of the target gene upstream or downstream the Cre gene in the receptor plasmid could affect the efficiency of the expression.