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.

Transformation Protocol

All the previously assembled BioBricks were transformed into DH5α competent cells acquired from New England Biolabs (NEB ®). In order to do so, NEB ®’s transformation protocol (3) was used: 50 μl of competent cells were added to microtubes; then, 5 μl of each previously assembled device (DNA concentration between 200 and 300 pg/ml, as determined by spectrophotometry) were pipetted into the tube, which was placed on ice for 30 minutes. Afterwards, the samples were submitted to 30 seconds of a 42°C heat shock; after which they were placed on ice for another 5 minutes. After incubation on ice, 950 μl of SOC medium were added to each mixture.
The tubes were placed at 37°C and 250 rpm for 60 minutes. Finally, 200 μl of each sample were plated into warm, solid LB media with 0.1% v/v of antibiotic (kanamycin 15 mg/ml for device 1, and chloramphenicol 35 mg/ml for devices 2, and 3).
After 12 hours of incubation at 37°C, a single colony was isolated from each plate and cultured overnight in liquid LB media with the previously stated concentration of antibiotic. A part of these liquid cultures was used for plasmid extraction and isolation; while the rest of them was used to perform relative fluorescence measurements. Both extraction and fluorescence measurement protocols are described later.

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