Team:Slovenia/METHODS and PARTS/protocols/bm

Fun fact:

Synthesis of violacein

Overnight cultures of E. colicontaining plasmids encoding zinc finger fusion proteins for violacein synthesis with or without plasmid encoding DNA program were diluted 1:1000 in fresh Luria Bertani broth and grown at 30°C in the presence of appropriate antibiotics. At various time points samples were taken. Bacteria were lysed by addition of equal volume of 10% SDS, and violacein extracted with 1:1 (v/v) ethylacetate. After brief vortexing, the organic phase was collected and absorbance spectrum (575 nm) was measured. To better separate and quantify the amount of violacein and deoxyviolacein, samples were further analyzed by HPTLC and mass spectroscopy. The quantity of violacein or deoxyviolacein production with or without DNA program or in the presence of scrambled DNA program was compared.

TLC

Thin-layer chromatography (TLC) is a type of liquid chromatography in which the stationary phase is the form of a layer on a glass, aluminum, or plastic support. TLC plates are usually developed by capillary flow of the developing solvent (mobile phase) without pressure in ascending or horizontal modes. Modern TLC, usually termed as high-performance thin-layer chromatography (HPTLC) can in combination with densitometry result in fast separations and quantitative results with accuracy and precision similar to HPLC and GC and enables a high-throughput screening. TLC compared to HPLC enables separation with much lower solvent consumption per sample.

Quantitative determination of violacein and deoxyviolacein in the samples (ethyl-acetate extracts) was performed by TLC on 10cm×20cm HPTLC silica gel 60 plates (Merck, Darmstadt, Germany). Solutions of violacein standard (0.01 mg/ml in methanol) and samples (ethyl-acetate extracts) were applied on the plates by means of Automatic TLC Sampler 4 (Camag, Muttenz, Switzerland). The plates were developed in 4 min simultaneously from both sides up to 4.5 cm from the bottom of the plate in dichloromethane:methanol (6:1, v/v) using a horizontal developing chamber (sandwich configuration). Quantitative determination of violacein and deoxyviolacein was performed by densitometry at 570 nm after scanning the spectra directly on the developed HPTLC plates. The quantity of violacein production with or without nucleic acid program or in the presence of scrambled nucleic acid program was compared. The identification of the studied compounds was also confirmed by the UV/VIS spectra scanned directly on the developed HPTLC plates and by mass spectra (LCQ mass spectrometer - electrospray ionization in a positive mode; Thermo Finnigan, San Jose, CA, USA) after on-line extraction from the HPTLC plates by means of Camag TLC-MS interface.

Synthesis of carotenoids

Overnight cultures of E. coli containing plasmids encoding zinc finger fusion protein for astaxantin synthesis with or without plasmid encoding DNA program were diluted 1:1000 in fresh Luria Bertani broth and grown at 30^oC in the presence of appropriate antibiotics. At various time points samples were taken and bacteria were removed by sedimentation (5 min at 13.000 rpm). To facilitate bacterial lysis, bacterial pellets were a freeze-thawed at -80^oC room temperature, respectively. Carotenoids were extracted with mixture of organic solvents.

HPLC

High-performance liquid chromatography (HPLC) is the most common separation technique for analysis, isolation and purification of various samples like pharmaceuticals, food and plant extracts, dyes etc. where compounds of the mixture are separated by the distribution between the stationary phase (silica gel, reverse phase etc.) which is packed into the stainless-steel column and the mobile phase under high pressure. The separation is followed by the detection, which can be performed by different types of detectors among which the UV/Vis and mass spectometers (MS) are the mostly used.

Samples of carotenoids were then analyzed by HPLC system consisted of LC pump P2000 (Thermo, Finnigan, San Jose, CA, USA), autosampler Spectrasystem TPS (AS 100) with fixed 20 µl loop and UV/Vis detector (Thermo, Finnigan). Separation was performed on a stainless-steel column Prontosil C30,(250x4.6 mm I.D.) with Phenomenex HPLC guard cartridge C18 as a precolumn using the following mobile phases: acetone for beta-carotene and lycopene; acetone:water (9:1, v/v) for zeaxanthin or canthaxanthin. All compounds were detected at 450 nm. Flow rate was 1 ml/min for all compounds while run time was 15 min for beta-carotene or lycopene and 25 min for zeaxanthin or canthaxanthin.