Resultados de simulación

3.1.1.

Working with bacteria

3.1.1.1. Cultivation of bacteria in liquid medium

A single bacterial colony was removed from a LB plate with a toothpick and inoculated in LB medium containing 100 µg/ml ampicillin. The bacteria culture was shaken at 160 rpm at 37°C for 12-16 h. Other selective medium were used depending on the existence and kind of plasmids inside the bacteria.

3.1.1.2. Streaking of bacteria plates5

Bacteria were picked from a frozen culture by immersing a loop or a toothpick in the cryo tube containing the culture. Alternatively bacteria were picked from a liquid culture by immersing a loop in it. The loop used for inoculation was flamed and briefly cooled before it contacted the colony. Then the loop was used to streak out the cells on the surface of LB plates. After an incubation of 12 -16 h at 37°C colonies were visible.

3.1.1.3. Freezing of bacteriacultures

0.8 ml of an over night culture in selective medium was transferred into cryo tubes, mixed with 0.2 ml glycerine and stored at -80°C.

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Every laboratory glass, plastic and metal ware used was sterile.

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Water refers to distilled water.

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3.1.1.4. Transformation of E.coli

Bacterial transformation is used to amplify plasmid DNA in a short time. 200 µl of

E.coli DH5α were thawed slowly on ice. Then, 20 µl of a ligation mixture (or 100 ng plasmid DNA) was added and the sample was incubated on ice for 20 min. After a heat shock for 2 min at 42°C the sample was placed into ice again. Subsequently 500 µl LB medium was added and cells were shaken for 1h at 37°C. After this time appropriate amounts were plated on LB medium containing ampicillin (2.7.1). Only cells containing the gene for resistance against ampicillin were able to grow up after incubation for 24 h at 37°C.

3.1.2.

Working with yeast

3.1.2.1. Culture of yeast cells in liquid medium

Two or three yeast colonies were inoculated in different media depending on the experiment. The cells were shaken at 200 rpm in plastic tubes or Erlenmeyer flasks at 30°C. All media and objects used for the culture were previously sterilized.

3.1.2.2. Freezing of yeast cells

Yeast cells were taken from a rich medium plate using an inoculating loop. Then cells were transferred to a cryo tube and mixed with 1 ml of freezing medium (2.8). Finally cells were stored at -80°C.

3.1.2.3. Determination of the yeast cell number

A Neubauer counting chamber and the suitable cover glass were cleaned with 70 % ethanol. The cover glass was set on the counting chamber, so that Newton rings were formed at the bearing surfaces. This indicates that the distance between glass and counting chamber is exactly 0.1 mm. A drop of the cell suspension was pipetted on the lower fissure between the cover glass and chamber.

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Then the cells of 25 small squares were counted under the microscope and the cell density of the suspension was determined according to the following equation:

104 = Volume of 25 small squares = 1mm2 surface x 0.1 mm depth = 0.1 mm3 = 10-4ml

3.1.2.4. Transformation of S. cerevisiae

Two or three single colonies of the yeast strains were inoculated in 10 ml YPAD and incubated ON under agitation at 30°C. Then 3 x 106 c/ml were inoculated in 100 ml of YPAD and shaken at 30°C till a cell concentration of at least 107 c/ml was reached (3 - 3.5 h). Cells were harvested by centrifugation, the supernatant was removed and the pellet was washed with 20 ml water. Subsequently cells were resuspended in 20 ml 0.1M LiOAc and shaken for 15 min. After centrifugation cells were resuspended in 0.1 M lithium acetate with a concentration of 109 c/ml. Per transformation 95 µl of the cell suspension were mixed (briefly vortexed) with 20 µl plasmid DNA, 240 µl PEG 50%, 31 µl 1M LiOAc and 15 µl ss salmon sperm, which previously was denatured at 95°C for 10 min. Cells were shaken for 30 min at 30°C before a heat shock at 42°C for 20 min. After centrifugation (5000 rpm, 1 min), control cells were resuspended in 1000 µl deionized water, thereof 50 µl were plated on selective medium. For transformation cells were resuspended in 500 µl and thereof 100 µl were plated on selective medium. Finally plates were incubated at 30°C for 72 h. Transformation rate was determined according to the following equation:

For the creation of new mutants the method differs as follow: After the heat shock and centrifugation cells are shaken 2 h in 1 ml rich medium at 30°C, then cells are harvested (5000 rpm, 1 min) and the pellet is resuspended in 400 µl milli-Q water. Finally 100 µl cells were plated on selective medium.

Transformation rate = colony number / µg DNA

3.1.2.5. Generation of knockout mutants by homologous recombination

Knockout mutants were produced by the complete substitution of chromosomal genes through selectable marker genes presented in plasmids or cassettes. These plasmids and cassettes include regions of homology with the target gene that allows their correct substitution by means of homologous recombination. The transformed cells were plated on selective medium and after three days cells replica were plated on fresh selective medium. Only the strains presenting the selective gene could grow on this medium. Finally the healthiest colonies of these plates were examined.