5 Materials and Methods
5.3 Functional Cloning
5.3.3 Cosmid characterization
5.3.3.1 Cosmid preparation from Leishmania culture
The cosmid DNA was isolated from the leishmanial cells by alkaline lysis and subsequent phenol- chloroform-extraction. Fifty ml of Leishmania culture were grown to high density (~2x107 cells/ml)
and cells harvested by centrifugation at 4 °C and 1260 g for 10 min. The cells were washed twice with PBS (pH 7.0) and were lysed with 1 ml of solution 1 and 2 ml of solution 2. After gentle shaking for 2- 5 min 1.5 ml of solution 3 were added and incubated on ice for 5-10 min. After centrifugation of this suspension at 4 °C and 3220 g for 30 min, the supernatant was filtered to exclude all cell particles, followed by an RNase digestion (40 µg/ml) at 37 °C for at least 30 min. For the extraction of the DNA, 1 volume of phenol: chloroform: isoamyl alcohol (25:24:1, v/v) was added, mixed and centrifuged at 20 °C and 3220 g for 4-6 min. The lower, organic phase was discarded. These steps were repeated twice, with the last repetition being modified by adding an equal volume of chloroform: isoamyl alcohol (24:1, v/v) and transferring the upper, aqueous phase into a fresh tube. The DNA was precipitated by the addition of 0.7 volumes of isopropanol and incubation at room temperature for 10 min. After the DNA was centrifuged to the bottom of the tube at the same conditions as before for 30 min, the supernatant was carefully discarded and the DNA pellet washed with ice-cold 70%
ethanol. The supernatant was removed thoroughly and the pellet dissolved in 20-40 µl of TE buffer (pH 8.0). The cosmid DNA was stored at 4 °C.
5.3.3.2 Transformation of E. coli with cosmid pcosTL
The cosmids, probably bearing the inserts with the genes responsible for the low MNNG tolerance, had to be amplified in E. coli.
Cells of the E. coli strain XL1-Blue were brought into a transformation competent state by serial washing steps of the cells with ice-cold 0.1 M CaCl2. Frozen cells were colonized on LB agar plates and
incubated overnight at 37 °C. The next day a pre-culture of 10 ml was inoculated with one colony and again incubated overnight at 130 rpm and 37 °C. A volume of 100 ml LB medium was inoculated with this overnight culture to an optical density of OD600=0.1 in a 500 ml flask. The cell density was
monitored photometrically and the growth was stopped at an OD600 of 0.6-0.7 by chilling the cells on
ice for 20 minutes. After a centrifugation step for 10 min at 4° C and 5000 g, the supernatant was discarded and the cell pellet resuspended in 10 ml ice-cold sterile 0.1 M CaCl2. These washing steps
were repeated with 4 ml ice-cold sterile 0.1 M CaCl2.The volume of the competent cells was
estimated and sterile glycerol was added to a final concentration of 10%. The cells were then aliquoted in 200 µl and either stored at 4 °C for use within the next 48 h or frozen in liquid nitrogen and stored at -80 °C until use. The transformation efficiency was tested for each lot using the plasmid pUC18.
For the transformation, the competent cells were thawed on ice and mixed with 1-5 ng of the cosmid preparation. This mix was placed on ice for 30 min and then incubated in a water bath of 42 °C for exactly 30 sec. After the heat shock, the cells were cooled on ice for 2 min, then suspended in 1 ml warm SOC medium without antibiotics and incubated at 37 °C and 250 rpm for 1 h. For the selection of successfully transformed cells, 200 µl of this culture were plated on an LB agar plate which contained 75 µg/ml ampicillin. The grown colonies were used for the downstream analyses. For long-time storage, 300 µl of overnight culture was mixed with 300 µl of 10% glycerol. This mix was frozen and kept in a cryotube at -80 °C.
5.3.3.3 Isolation of cosmid DNA from E. coli
One colony was used to inoculate an LB agar plate (+ampicillin) and 4 ml of liquid LB medium. Both were incubated overnight at 37 °C. The agar plate was stored at 4 °C as back-up, the cells grown in liquid medium were harvested by centrifugation at 4 °C and 1400 g for 15-30 min. They were resuspended in 200 µl of solution 1 (for recipes see chapter 5.1.3) transferred to an Eppendorf tube and mixed with 400 µl of solution 2 by inverting the tube several times and incubating at room
temperature for 3 min. Upon addition of 300 µl of solution 3, the tubes were incubated for 5 min on ice, and then centrifuged at 4 °C and 13000 g for 30-60 min. The supernatant was mixed thoroughly with 1 ml of isopropanol by inverting the tube several times to precipitate the DNA. After an additional centrifugation step under the same conditions, the DNA pellet was washed with 700 µl 70% ethanol. The dried DNA pellet was suspended in 20 µl TE/RNase (20 µg/ml) and incubated at 37 °C for up to 120 min. The DNA was stored at 4 °C.
5.3.3.4 Restriction analysis of the recombinant cosmids
The cosmids were subjected to restriction analyses in order to identify differences in the fragment patterns due to the presence of different inserts in the cosmid clones. One reaction consisted of 3- 4 µl DNA, 20 U EcoRV and XbaI in a total volume of 15 µl. The buffer and BSA were used according to the manufacturer´s instructions. The reaction was incubated for 15 minutes at 37 °C.
The cosmid fragments were analysed by electrophoresis. Each resulting fragment pattern contained two fragments originating from the cosmid backbone at 7422 and 815 bp. The electrophoresis was carried out at ~100 V for 1.5-2 h in a 0.7% agarose gel. A special agarose (SeaKem LE agarose) was used for the separation of large fragments. The DNA was visualized after soaking the gel into a staining bath containing 100 µl ethidium bromide (10 mg/ml) per 2 l aqua dest. for 20 min. For estimation of the fragments´ sizes, the 1kb DNA Extension ladder was used.
5.3.3.5 Identification of the cosmid inserts in positive clones
The cosmid inserts were partially sequenced to identify their sequences for those clones that exhibited low mutation tolerance in the presence of MNNG. The primers to initiate the sequencing reaction from both ends of the insert were complementary to the cosmid sequence near the ligation sites. Their sequences were 5´-CCGTGTGATATCAGATGCCC-3´ (CH2) and 5´-
GGAAACAGCTATGACCATG-3´ (M13rev). The resulting DNA sequences each contained a part of the insert. They were then used for a BLAST search using the NCBI database to determine the start and end points of the insert. All available sequences of different Leishmania species were used for the alignments. VectorNTI Advance 11 and BioEdit 7.0.9.1 [100] were used for the handling of the sequencing data.