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.

MiniPrep Protocol

Common miniprep plasmid-DNA extraction was performed. To do so, an isolated colony obtained from the transformation step was transferred from the Petri dish into an Erlenmeyer flask containing LB medium with the selection antibiotic (0.1% v/v). The flask was incubated overnight at 37°C and 250 rpm; 10 ml of the resulting culture were centrifuged at 13500 rpm for 30 seconds, so that biomass could be separated. The supernatant was discarded and cells were resuspended in 350 μl of STET buffer. The mixture was then transferred to a 1.5 ml microtube, where 5 μl of lysozyme (10 mg/ml) were added. The mixture was incubated during 3 minutes, after which the tube was transferred to a boiling water bath for 2 minutes in order to inactivate the enzyme.
Afterwards, the sample was centrifuged at 13500 rpm for 10 minutes. The bacterial pellet was taken out of the liquid using a sterile micropipette, and 10 μl of RNase A were added. The mixture was incubated for 10 minutes at room temperature. Then, 20 μl of sodium acetate (3M), and 250 μl of isopropanol were added. The mixture was gently stirred and incubated for 10 minutes at room temperature. Afterwards, it was centrifuged for 10 minutes at 12400 rpm; the supernatant was discarded, and the pellet washed 2 times with 1 ml of ethanol 70% v/v. Finally, the DNA was resuspended in 100 μl of distilled water and quantified by spectrophotometry.

Assembly Protocol

Promoters from New device 1 and 2 (contained in a psB1C3 plasmid),were digested using the restriction enzymes SpeI and EcoRI. Reagents were added to a 0.5 ml PCR tube in the following order: 12.5 μl of water for molecular biology, 4 μl of NEB® Buffer 2.1, 0.5 μl of BSA, 20 μl of DNA (BioBrick BBa_K823005 in psB3K3 backbone), 1.5 μl of SpeI enzyme, and 1.5 μl of EcoRI enzyme. The content of the tube was gently mixed, and placed at a Thermoblock at 37°C for 75 minutes. After incubation, the tube was placed at a water bath at 80°C for 20 minutes so that the enzyme could be inactivated. Finally, the digestion product was stored at -20°C.

The GFP cassette, BBa_E0240 (contained in a psB1C3 plasmid) was also obtained by digestion, now using XbaI, and PstI. The same procedure was used, but now 20 μl of DNA (BioBrick BBa_K823005), 1.5 μl of XbaI enzyme, and 1.5 μl of PstI enzyme were added to the tube. The digestion product was also stored at -20°C.

This same protocol was followed to obtain the desired backbone (psB1C3) by digesting a RFP-containing psB1C3 plasmid, using the restriction enzymes EcoRI and PstI; the product was stored at -20°C.

Then, ligation of the 3 previously obtained DNA fragments was then performed; since only one possible combination for BioBrick ligation existed (apart from relegation of two backbones) given the sequence of the cohesive ends generated by the restriction enzymes, there was no need to previously purify the DNA.
To ligate, 2 μl of water for molecular biology, 2 μl of NEB® T4 Ligase buffer, 8 μl of the first digestion product (BBa_K823005), 4 μl of the second digestion product (BBa_E0240), 5 μl of the third digestion product (psB1C3 backbone), and 1 μl of NEB® T4 Ligase were added to a 0.5 ml PCR tube. The mixture was incubated at 16°C for 24 hours, then inactivated for 10 minutes at 65°C and finally stored at -20°C.

Fluorescence Measurement Protocol

In order to measure the relative fluorescence of the three different GFP-expressing genetic devices, a fluorometer was used. This was done at the Research Center CINVESTAV (Center of Advanced Studies and Investigations) of National Polytechnic Institute, at Mexico City. Firstly, isolated colonies of bacteria expressing BioBricks BBa_I20260 (Device 1), BBa_K823005 + BBa_E0240 (Device 2), and BBa_K823012 + BBa_E0240 (Device 3); as well as common Top10, and DH5α strains (negative controls) were cultured overnight at 37°C in a Shaker using 5 ml of LB media with 0.1% v/v of antibiotic (15 mg/ml kanamycin for device 1, and 35 mg/ml chloramphenicol for devices 2 and 3). Then, 1 ml of the resulting culture was further subcultured 2 hours at 37°C and 250 rpm in another 5 ml of LB media, with no antibiotic; while the rest of it was kept at 4°C.
After two hours, both sets of samples (stored at 37°C and 4°C respectively) were centrifuged at 13000 rpm for 1 minute to separate the biomass. As suggested in previous fluorometric studies (4), the supernatant was discarded, and the samples washed with 1 ml of PBS 1X three times using the same centrifuge conditions, so that all the remnants of LB liquid media could be removed (because LB is capable of emitting fluorescence). Finally, the sample was resuspended in 1 ml of PBS and diluted, the absorbance of 100 μl of the sample was determined at a wavelength of 600 nm using a spectrophotometer. Using the optical density data obtained, the original samples were serially diluted to create a standard curve with values of OD600 ranging from 0 to 1, with increasing intervals of 0.2 units. This data was later translated to cell number.
After verifying the OD600 of each dilution, samples of 150 μl for each biomass concentration were transferred to a fluorometer plaque, and their relative fluorescence was measured by triplicate using the appropriate filters (excitation 485 nm, emission 510 nm), while stirring. A standard curve was obtained for each bacterial strain (DH5α, Top10, and devices 1 to 3) using the statistical mean of the 3 measurements.

In order to prove that BioBrick assembly and transformation had been properly done, a restriction map was generated. This was done by extracting, and digesting (as explained previously) each BioBrick, as well as each fully assembled device, with a unique combination of restriction enzymes. The resulting DNA fragments were analysed by DNA electrophoresis in 0.8% agarose gel, at a voltage of 110 V.
The results of this restriction analysis are shown in figure 1, where all electrophoresis results photographs are superposed in a single image, so that fragment sizes can be compared.
Device 1 (BBa_I20260) was digested using XbaI, PstI, and the combination of both. Device 2, using enzyme NcoI, and Device 3, with enzymes EcoRI and PstI acting together. Control samples with no enzyme are also included.
In order to compare fragment sizes, individual BioBricks were also digested. Promoter BBa_K823012, contained in backbone psB1C3 was digested using EcoRI and SpeI; and mutant GFP gene, BBa_E0240 (also contained in psB1C3 plasmid backbone) was digested with XbaI and PstI.

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