"
Team:USP-UNESP-Brazil/Project2
From 2012.igem.org
| Home | Team | Official Team Profile | Projects | Parts Submitted to the Registry | Modeling | Notebook | Safety | Attributions |
|---|
Contents |
Overall project
Plug&Play Plasmid
The Plug&Play machine propose aims to construct a tool for a faster protein expression, what would allow to accelerate the choice of genes of interest. The main idea is to create an open source set of genes for various expression cases, homologous to the different expression vectors available on the market. As proof of concept, is proposed the genesis of a plasmid that could allow the expression of any protein inside E.coli in two passages: PCR (Polimerase Chain Reaction) and transformation.
The main concept which the project relies on is the Cre recombinase protein action. This enzyme can accelerate the recombination between specific sites known as loxP, whose sequence have two ligation sites allowing recombination inside the chromosomes. By this action the Plug&Play system is composed of a pair of primers to amplify the ORF sequence of a given protein. Each set of primers have the sequence capable of recognize the ORF flanked by one of the assembling sites of loxP site. This assembling sequences will have two main functions: circularize the PCR product on the moment it enters on the bacteria and the recombine with a new plasmid that will be inside the bacteria. This plasmid is put in the bacteria, allowing it to accept any PCR amplified ORF flaked by loxP sites, using the recombination mechanism (Cre recombinase) to insert the sequence on the right place, allowing to the gene to become susceptible to the transcription machinery
