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 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.

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.

The Plug&Play system consist of two parts;

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.

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.

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.

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.