ORIGENES DE DONDE SURGE LA OBLIGACION ALIMENTARIA

2.2.1.1 Extraction of plasmid DNA from bacteria

For small scale production of DNA up to 25 |ig (minipreparation), 2 ml of LB containing

0 . 1 mg/ml ampicillin was inoculated with a single colony from the agar plate and

incubated overnight with shaking at 37°C. Plasmid DNA was prepared using the Wizard miniprep kit (Promega) which employs the alkaline lysis method of (Birnboim, 1983). Bacterial cells were pelleted at 10 000 g for 1 min and resuspended in 200 pi of the cell resuspension buffer (50 mM Tris-HCl pH 7.5, 10 mM EDTA, lOOmg/ml RNase A). For cell lysis and precipitation of the proteins, 200 pi of cell lysis solution (0.2 M NaOH, 1% SDS) and 200 pi of neutralization mixture (1.32 M potassium acetate pH 4.8) was added after 1 min of incubation and the tubes gently inverted. Cell debris and proteins were then removed by pelleting for 5 min at 10 000 g. The supernatant was collected and incubated with 1 ml of a DNA binding resin for 1 min at room temperature. The resin with bound plasmid DNA was then separated from the solution by using a spin minicolumn. After washing the column once with wash buffer containing ethanol, the DNA was eluted from the beads with 50 pi water by spinning the minicolumn for 20 s at 10 000 g. The resin

stayed inside the minicolumn and the water containing the DNA was collected into an eppendorf tube.

The O^iagen maxi kit ( Gtiagen, Crawley, UK) was used for the preparation of DNA on a large scale (up to 500 pg) using 400 ml of bacterial culture. The Q. iagen protocol is based on the modified alkaline lysis procedure o f (Birnboim, 1983). The procedure follows the same principle as the Wizard kit. After lysis under alkaline conditions using the solutions provided, the lysate was filtered and applied to a equilibrated Qiiagen column containing a resin which selectively binds DNA while allowing proteins, RNA and cell debris to flow through. The column was washed once with the wash buffer provided and DNA eluted with the elution buffer. The DNA was precipitated by addition o f 0.7 volumes of isopropanol followed by centrifugation at 10 000 g for 30 min to pellet the DNA. The pellet was then air-dried, resuspended in 300 pi water and the DNA concentation measured by spectrophotometry. Mormahy a DNA concentration of

1^5l|itto»sobtained.

2.2.1.2 Determination of DNA concentration

The concentration o f nucleic acid in aqueous solutions was determ ined by spectrophotometry. An OD (optical density) of 1 at 260 nm was taken to be equivalent to 50 pg/ml DNA. The amount of DNA could also be estimated by loading 1 pg of DNA marker (1 Kb DNA ladder, Gibco BRL, Life Technologies, Paisley, UK) and comparing the intensity of the standard bands with the sample.

2.2.1.3 Restriction enzyme digestion of DNA

Plasmid DNA was digested using 10 units enzyme/pg DNA for 1-2 h. The restriction enzymes EcoRI, P a d , BamHI, Notl were supplied from Roche Molecular Biochemicals, Lewes, UK and New England Biolabs, Hitchin, UK. The digestionvo«iScarried out in the appropiate buffer as recommended by the supplier. To determine if complete digestion had occurred a small aliquot was loaded onto a gel and when possible compared to an undigested sample.

2.2.1.4 Agarose gel electrophoresis of DNA

DNA was fractionated according to size on agarose gels using a flat bed apparatus. Gels contained 0.8% -2.0% w/v agarose (depending on the size of the DNA fragments to be separated) in TAE buffer and ethidium bromide added to 0.5 pg/ml and were run in TAE buffer. A 1:10 dilution o f 10 x loading buffer was added to the samples before loading

into the wells. Gels were run at 7 volts/cm for as long as required to get adequate separation o f DNA fragments (0.5 to 2 h). DNA was visualized by illumination with short wave (254 nm) ultraviolet light.

2.2.1.5 Isolation of DNA fragments from agarose gels

The band o f interest was excised from the agarose gel and the DNA extracted using the Jetsorb kit (AMS Biotechnology, Witney, UK) following the manufacturers instructions. The protocol is based on the method of (Vogelstein and Gillespie, 1979) and makes use o f the fact that DNA binds to glass. In short, the agarose is dissolved in a solution containing concentrated NaClO^ and the DNA extracted using the silica bead suspension provided.

2.2.1.6 Ligation of DNA fragments into vectors

Ligations were carried out in a total volume of 15 |Ltl containing vector and insert DNA (50 ng vector with a 3 times molar excess o f insert), 0.1 volumes of 10 x ligation buffer (300 mM Tris-HCl pH7.8, 100 mM MgClz, 100 mM DTT, 10 mM ATP) and 10 units of T4 DNA ligase (Promega, Southhampton, UK). The mixture was incubated for 2 h at room temperature or for 18 h at 16°C. To determine vector religation, a control ligation was set up where the insert was replaced with water.

V ector DNA fragments to be used for ligation were dephosphorylated following restriction enzyme digestion to remove 5 ’ terminal phosphate groups in order to prevent vector religation using shrim p alkaline phosphatase (A m ersham , Life Science, Cleaveland, USA). The digest was further incubated at 37 °C for 45 min and then loaded directly onto an agarose gel to isolate the vector fragment and to remove enzymes.

2.2.1.7 Transformation o f bacteria

2 |xl o f ligation mix or 10 ng of plasmid DNA was added to 20 |xl o f competent bacteria (XL-1 Blue, Stratagene, Cambridge, UK) and left on ice for 30 min. The bacteria were heat shocked at 45°C for 45 s, then put onto ice for 2 min. 80 p.1 of LBroth were added and the cells incubated at 37°C for 45 min before plating on LB plates containing 0.1 mg/ml ampicillin. The plates were incubated overnight at 37 °C. A control transformation where the insert DNA was replaced by water in the ligation mix was also transformed and plated out to estimate the background level of vector religation. Colonies were screened by preparing plasmid DNA which was then subjected to restriction digest analysis to determine the presence and orientation of the insert.

2.2.2 Preparation, expression and analysis of proteins