3 Materials and Methods
3.2 Media, Strains and Plasmids
3.2.1 Media
Within the framework of this project, various media were used to cultivate both bacteria and fungi. During the medium preparation process, all of these were autoclaved for 20 min at 121°C.
For cultivation of bacteria, both LB- and NZY media were used:
Table 3-3 LB Medium pH 7.5* (Luria et al. 1960; Sambrook et al. 1989) Contents Weight/
Volume Manufacturer
Bacto-Trypton 10.0 g/l Difco Laboratories, Detroit, MI, USA
Yeast extract 5.0 g/l Select Yeast Extract Sigma Aldrich Chemie GmbH, Steinheim, D
NaCl 10.0 g/l C. Roth & Co. GmbH, Karlsruhe, D
*The pH of the medium was adjusted to pH 7.5 with 5 M NaOH, before being autoclaved.
LB medium was first described in the early days of phage genetics studies (Luria et al. 1960) and is still widely used by the molecular biologist today. All of the formulations contained 10 g tryptone and 5 g yeast extract/liter. Some also included 0.1% sugar, which is not used in this case. NaCl amounts can vary between 0.5 g and 10 g. For E. coli growth within the framework of this thesis, 10.0 g NaCl per liter were used.
To prepare the LB agar plates used during this thesis, 18 g/l Agar-Agar was added to freshly prepared (not autoclaved) LB medium and autoclaved. If a selection for antibiotic resistance of transformed bacteria was required, one of the following antibiotics was added to the LB agar after autoclaving:
• Ampicilline 100 µg/ml
• Kanamycine 50 µg/ml
The medium was then poured into Petri dishes and stored at 4°C.
Table 3-4 NZYM (NZY) pH 7.5 (Blattner et al. 1977; Sambrook et al. 1989) Contents Weight/
Volume Manufacturer
NZ-Amine 10.0 g/l Sigma Aldrich Chemie GmbH, Steinheim, D
Yeast extract 5.0 g/l Select Yeast Extract Sigma Aldrich Chemie GmbH, Steinheim, D
NaCl 5.0 g/l C. Roth & Co. GmbH, Karlsruhe, D
Above ingredients were autoclaved for 20 min at 121°C, before addition of 2 M MgSO4 4.0 ml/l
Traditionally, the E. coli host for lambda phages is grown on a NZY medium. This medium is not as rich as LB medium, and E. coli growth is slower, making it less likely that E. coli will overgrow the phage.
During this thesis, NZY medium was used to prepare TopAgar for phage lysates. TopAgar was prepared as follows: 0.7%, agarose (Carl Roth GmbH + Co, Karlsruhe, D) was mixed with NZY-medium and autoclaved. If required, TopAgar was reheated several times in the microwave oven. For a description of the subsequent plating procedure, see chapter 3.5.
For cultivation of all fungi besides Botrytis fabae and Ascochyta fabae, malt extract was used:
Table 3-5 Malt Extract
Contents Weight/ Volume Manufacturer
Malt extract 30.0 g/l Biomalt, Gesundprodukte, Kirn D
Malt extract is a rich medium for fungal growth and stock culture preparation. If not otherwise mentioned, all fungi used for further DNA preparation were grown using malt extract in liquid culture (20°C, 80 rpm, up to one week). Malt extract was also used for infiltration experiments with Botrytis fabae in a dilution of 1/1’000 to stimulate pathogenesis of this fungus.
The cultivation of B. fabae and A. fabae took place using oatmeal- and potato-dextrose liquid culture medium, as well as hay extract:
Table 3-6 Potato Dextrose Liquid Culture Medium (MacFaddin 1985) Contents Weight/ Volume Manufacturer
Potato Dextrose Broth
24 g/l DIFCO Laboratories, Detroit, MI, U.S.A
Table 3-7 Oatmeal Liquid Culture Medium Contents Weight/
Volume Procedure
Oatmeal 30 g/l Freshly ground, organically grown oatmeal was cooked for about 0.5 h in tap water. The liquid phase was then decanted and autoclaved for 20 min at 121°C
Table 3-8 Hay Extract Contents Weight/
Volume Procedure
Hay 30 g/l Dry hay was cooked for about 1 h, then autoclaved for 20 min at 121°C for a better extraction, filtered, replenished to 1’000 ml and again autoclaved for 20 min at 121°C.
Botrytis fabae was also cultivated on Oatmeal agar or Hay agar. To create Oatmeal agar, the above described oatmeal liquid culture medium was mixed with 12 g/l agar after cooling. The agar was then autoclaved for 20 min at 121°C. To make Hay agar, the prepared hay-extract was mixed with 15g/l agar after cooling. The agar was then also autoclaved for 20 min at 121°C.
Oatmeal- and Hay agar were used to stimulate the spore formation of Ascochyta fabae and Botrytis fabae. Whereas Hay agar is a minimal medium, Oatmeal agar provides more nutrition, especially in the form of starch.
The yeast S. cerevisiae, strain RE700A (see Table 3-12) used during this thesis was cultivated in an SC-Maltose medium with uracil. If the yeast had been transformed with the vector pDR195, the cultivation medium did not contain uracil:
Table 3-9 SC-Maltose Medium (Shermann 1991) Contents Weight/
Volume Manufacturer Yeast Nitrogen
Base
6.8 g/l Difco Laboratories, Detroit, MI, USA Maltose 20.0 g/l Merck, Darmstadt, D
Dropout mix 2.0 g/l See Table 3-10
The SC-maltose medium was used to prepare SC-maltose agar plates. For this, 17 g Bacto-agar were added to 500 ml aqua bi-distilled in a one-liter flask. The other ingredients were put into a separate flask and filled up to 500 ml with aqua bi-distilled. Both flasks were autoclaved separately, and after having been allowed to cool to approximately 80°C, both solutions were mixed together and poured into Petri dishes.
Table 3-10 Dropout Amino Acid Mix for SC-Maltose Medium
Contents Weight Contents Weight
Adenine 0.5 g Alanine 2.0 g
Arginine 2.0 g Asparagine 2.0 g
Aspartarte 2.0 g Cysteine 2.0 g
Glutamine 2.0 g Glutamate 2.0 g
Glycine 2.0 g Histidine 2.0 g
Isoleucine 2.0 g Leucine 2.0 g
Lysine 2.0 g Methionine 2.0 g
Para-aminobezoate 0.2 g Phenylalanine 2.0 g
Proline 2.0 g Serine 2.0 g
Threonine 2.0 g Tryptophane 2.0 g
Thyrosine 2.0 g Valine 2.0 g
This dropout mix does not contain uracil. For the SC-Maltose + Uracil Medium, the dropout mix additionally contains 2.0 g uracil.
3.2.2 Strains
The following bacterial- and yeast strains were used during this thesis:
Table 3-11 Bacterial Strains TG1 (E. coli) SupE hsd∆ thi∆(lac-mo AB)[ F’(traD36, pro
AB, +laclq, lacZ ∆M15)]
(Sambrook et al. 1989)
For recombinant protein production, the Escherichia coli strain BL21 (DE3) plys E was used.
This strain descends from E. coli B and is deficient in both lon- and the outer membrane ompT proteases. Cells deficient of these proteases accumulate recombinant proteins at a high rate and are less likely to degrade these proteins during purification.
In order to obtain the sequence information for the EST project (see chapter 3.5), the E. coli strain NM514 was infected with λGT10 containing the Uromyces fabae gene bank.
For the production of plasmids for further transformation, the E. coli strain TG1 was used (see chapter 3.7.1).
In addition to the bacteria strains, the following yeast strain was transformed with pDR195, which contained the sequences of the PIGs to be studied (see chapter 3.7.2 and Table 3-13), and used to produce the PIG-proteins.
Table 3-12 Yeast Strain
Name Reference
RE700A (S. cerevisiae) (Reifenberger et al. 1995)
3.2.3 Phages and Vectors
The basic data for the EST project was obtained from a cDNA library of haustorial genes (Hahn and Mendgen 1997) kindly provided for use within the framework of this thesis by M.
Hahn. The library was created using a TimeSaver cDNA synthesis kit (Amersham Bioscience Europe GmbH, Freiburg, D), and the λGT10 phage.
The production of recombinant proteins for antibody production and the testing of PIGp signal sequences was realized using two different plasmid vectors, which are described in more detail in chapter 3.6:
Table 3-13 Plasmid Vectors
Vector Source/ Reference Selection Promotor
pET28a+ Novagen, CNBiosciences, Inc., San Diego, CA, USA (Studier and Moffatt 1986)
Kanamycine T7φ
pDR195 (Rentsch et al. 1995) Ampicilline PMA1