Overview

During 2014 iGEM competition, teams were requested to analyse the efficiency of 3 different genetic devices (BioBricks) using GFP as a marker of gene expression. Here, iGEM ITESM CEM team presents the results of this interlab fluorescence measurement study.

The three devices analysed are composed by a variable promoter, a gene encoding a mutant Green Fluorescence Protein (GFP) used as a marker of expression, and a plasmid backbone. Two promoters (BBa_J23101 and BBa_J23115, recently renamed BBa_K823005 and BBa_K823012 at iGEM’s catalogue) are used, both of them being members of a family of constitutive promoters described by Chris Anderson, member of iGEM Berkley Team, in 2006 (1). This family of parts is registered at the catalogue under the alphanumeric codes BBa_J23100 – BBa_J23119.

Two different plasmid backbones are used: a low-copy (psB3K3) and a high-copy plasmid (psB1C3). The GFP-expressing BioBrick remains the same for all devices (registered at the catalog as BBa_E0240), and is composed of a ribosome binding site (RBS), a mutant GFP gene, and two termination sequences.

The aim of this study is to report the relative efficiency of the following genetic devices:

1) Promoter BBa_K823005 in low-copy plasmid psB3K3
2) Promoter BBa_K823005 in high-copy plasmig psB1C3
3) Promoter BBa_K823012 in high-copy plasmid psB1C3

In order to do so, GFP (BBa_E0240) is used as a marker of gene expression or reporter gene, because of the ease of fluorescence measurement experiments.

GFP has long been used as a reporter of patterns of gene expression in both prokaryotes, were it is useful for characterization of promoters, enhancers and terminators; and in eukaryotes, were tissue-specific or time-specific gene expression can be traced (2). The basis of this procedure is the usage of GFP’s fluorescence as a reporter of activity of promoters and enhancers; the relative fluorescence of cells at different experimental conditions can be compared with statistical techniques, and so the efficiency of the parts can be tested.