All molecular and microbiological methods in this chapter have been performed according to either Sambrock et al. (1989) or Ausubel et al. (1987), unless otherwise stated.
4.2.1 Preparation of competent E. coli cells
Preparation of competent E. coli cells was done according to Inoue et al. (1990). A 5 ml overnight culture of the respective cells was inoculated directly from a respective agar plate. On the next day, 125 ml of LB-media was inoculated with the culture and incubated shaking at 180 rpm and 37 °C until the culture reached OD600 = 0.4-0.75. Culture was transferred to sterile 50 ml-falcon tubes and incubated on ice for 10 min. Afterwards, cells were centrifuged for 10 min at 4 °C and 1000 x g.
Supernatant was removed and pellet was resuspended in 20 ml TFB-buffer per falcon. After 10 min of incubation on ice, falcons were centrifuged for 10 min at 4 °C and 1000 x g again. Supernatant was removed and the pellets were resuspended in 4 ml of TFB-buffer and combined in one falcon. After the addition of DMSO to a final concentration of 7 % (v/v), cells were further incubated on ice for 10 min. Finally, cells were divided in 200 µl-aliquots and flash-frozen in liquid nitrogen. Aliquots were stored at -80 °C.
TFB-buffer
amount final concentration component
0.605 g 10 mM PIPES
0.441 g 15 mM CaCl2 x 2H20
3.728 g 250 mM KCl
adjust pH to 6.7
2.18 g 55 mM MnCl2 x 4H20
4.2.2 Heat shock transformation of E. coli
Heat-shock transformation of E. coli was done according to Mandel and Higa (1970). Competent E. coli cells were mixed with DNA in a maximal ratio of 10:1 (v/v). Cell-DNA mixture was incubated on ice for 20 min. Afterwards, a heat-shock was performed at 42 °C for 45 sec. After further incubation on ice for two minutes, 600 µl of LB medium was added. Sample was incubated at 37 °C for 30 min in case of plasmids harbouring an ampicillin resistance and for 90 min in case of plasmids harbouring a kanamycin resistance. In case of retransformations, 100 µl of the sample was plated out on LB-plates containing the respective antibiotics. In all other cases, the complete sample was pelleted via centrifugation, supernatant was discarded and remaining pellet was plated out. Plates were incubated over night at 37 °C.
4.2.3 Preparation of competent S. cerevisiae cells
Preparation of competent S. cerevisiae cells was done according to Ito et al., a method which was further refined by Gietz and Schiestl (Gietz and Schiestl, 2007; Ito et al., 1983). For preparation of competent yeast cells, 50 ml of YPD medium was inoculated with a single colony of the respective strain and incubated over night at 30 °C and 180 rpm. Five millilitres of this culture were used to further inoculate 100 ml of YPD medium. The resulting culture was grown at 30 °C for approximately 4 h to an OD600 of 0.6-0.7. Cells were pelleted via centrifugation at 4000 x g for 5 min and washed with 20 ml solution A. Afterwards, cells were resuspended in 4 ml solution A. Subsequently, 100 µl of a 10 mg/ml solution of heat-denaturated sperm-DNA and 100 µl of a 1 M histamine solution were added and mixed. Cells were divided in 200 µl aliquots and frozen at -70 °C.
Solution A
amount component
1 M sorbit
10 mM tricine
3 % (w/v) ethylene glycol
4.2.4 Transformation of S. cerevisiae
Transformation of S. cerevisiae was done according to Gietz and Schiestl (2007) and Ito et al. (1983).
For transformations, 5 µg of plasmid-DNA was added to a 200 µl aliquot of frozen S. cerevisiae cells.
Sample was incubated in a thermoshaker at medium speed and 37 °C for 5 min. Afterwards, cells were diluted in 1.2 ml solution B and further incubated at 30 °C for 1 h. Subsequently, cells were pelleted via centrifugation at 4000 x g for 1 min and washed three times with solution C. Cells were
METHODS PCRs were done using the Phusion-DNA polymerase (Finnzymes, Finland). Sample compositions and temperature gradients were done as follows: desired DNA-fragment in a respective vector. A PCR with primers specific for the respective vector was performed on the basis of whole cells as DNA-templates. A single E. coli colony was picked from an agar plate with a sterile toothpick. The cells were resuspended in a single PCR-sample and furthermore spread out on a LB-Agar plate supplemented with respective antibiotics for later use.
The PCR sample was composed as described in 4.2.5, except for that GoTaq-DNA Polymerase (Promega, USA) was used. The temperature-gradient was as follows:
94 °C 4 min
4.2.7 Overlap extension PCR
Domain swap variants of MmAWAT2 and MmDGAT2, as described in 5.3.4, were created by overlap extension PCR according to Horton et al. (1989). Respective fragments were at first amplified from wild type sequences. All primers for overlap extension PCR were designed with a 20 bp-overhang corresponding to the adjacent fragment in case of forward-primers and a 15 bp overhang in case of reverse primers. Fragments were purified via gel-extraction and eluted in 30 µl H2O. Of this eluate, 2 µl of the respective shorter and 3 µl of the respective longer fragment were used for overlap extension PCR using a standard PCR protocol and the Phusion DNA-Polymerase (Finnzymes, Finland).
4.2.8 Quick-Change PCR
All amino acid exchange variants used in this study were created by site directed mutagenesis (Hutchison et al., 1978). Respective primers listed in Table 9 were designed as follows: ~10 bases in direction of 5’-end of the mutation, ~20 bases in direction of 3’-end of the mutation, ~10 non-overlapping bases at the 3‘-end of both primers. Phusion DNA polymerase (Finnzymes, Finland) was used to ensure correct amplification. Temperature gradient and sample composition was used as follows:
amount component 0.5 µl dNTP (each 10 µM) 0.5 µl Primer A (10 µM) 0.5 µl Primer B (10 µM)
~ 3 ng DNA template 1 U DNA polymerase ad 50 µl H20
94 °C 4 min
18 x
{
94 °C 1 min 55 °C 30 sec.
72 °C 1 min / kb 72 °C 10 min.
After PCR amplification of the respective plasmid, sample was incubated with 10 U of DpnI at 37 °C over night in order to eliminate methylated template-DNA.
4.2.9 Restriction of DNA
DNA restriction was performed by utilization of DNA-restriction endonucleases, as reviewed by Boyer (1971) in the buffer recommended by the respective manufacturer and according to the manufacturer’s protocol.
METHODS
4.2.10 Ligation of DNA
Ligation of DNA was done by utilization of the T4-ligase (Weiss and Richardson, 1967) according to the instructions provided by the manufacturer.
4.2.11 Plasmid preparation from E. coli
For plasmid preparation, a respective 2 ml E. coli overnight-culture was inoculated with a single colony and supplemented with required antibiotics. On the next day, plasmid-DNA was prepared using the NucleoSpin™ Plasmid-Kit (Table 4) according to the instructions provided by the manufacturer. The procedure is based on a publication of Birnboim and Doly (1979).
4.2.12 Separation of DNA by agarose gel-electrophoresis
This method is based on a principle published by Aaij and Borst (1972). DNA-samples were mixed with an appropriate volume of 6x loading dye. Preparation of agarose-gels was done by boiling 1 % agarose (w/v) in TAE-buffer. Electrophoresis was performed for approximately 20 min, until the bromophenol-blue running front left the gel. Afterwards, gel was incubated in TAE supplemented with 2 µg/ml of ethidium bromide. DNA was visualised in UV-light.
TAE-buffer
amount component 40 mM Tris/HCl, pH 7.0 20 mM acetic acid
1 mM EDTA
6x loading dye
amount component 40 mM Tris/acetate pH 8.5
100 mM EDTA
0.1 % (w/v) SDS 50 % (v/v) glycerol
0.25 % (w/v) xylencyanol blue 0.25 % (w/v) bromophenol blue
4.2.13 Cultivation of E. coli Lemo21 (DE3) for protein expression screenings
Lemo21 (DE3) cells were used to screen for optimal expression conditions for pMAL_MmFAR1 and pGEX_DmFAR. Lemo21 cells contain a plasmid which codes for the natural T7 RNA polymerase inhibitor T7 lysozyme, which is expressed from a titratable rhamnose promoter. It is thus possible to adjust the level of T7 RNA polymerase in order to control expression conditions.
For screening experiments, two times 10 ml expression cultures were supplemented with either 0, 100, 250, 500, 750, 1000 or 2000 µM of L-rhamnose, appropriate antibiotics and 200 µl of an overnight culture. Cultures were grown at 30 °C and 180 rpm to an OD600 of 0.4-0.8. One of each culture was than induced with 400 µM IPTG and further incubated at 30 °C and 180 rpm for four
additional hours. Afterwards, OD600 of the culture was measured. Samples were taken according to the formula [0.5/OD600 = ml sample]. Samples were pelleted, resuspended in 50 µl 1x Laemmli buffer and incubated at 95 °C for 5 min. 10 µl of the samples were applied on a SDS-gel.
4.2.14 Cultivation of E. coli expression cultures for protein purification
For cultivation of E. coli expression cultures, 50 ml of 2xYT-medium supplemented with the appropriate antibiotics was inoculated with a single colony and incubated shaking at 200 rpm over night at 37 °C. On the next day, 1 l of 2xYT-medium, supplemented with appropriate antibiotics, was inoculated with 50 ml of an overnight culture. Culture was incubated shaking in 2 l-flasks at 200 rpm and 37 °C for 60-120 min, until the culture reached an OD600 of 0.4-0.8. Protein expression was induced by the addition of 400 µM IPTG. The culture was further incubated at 200 rpm and 16 °C for
~62 h, unless stated otherwise. Cultures were harvested by centrifugation at 3000 x g and resulting pellets were flash frozen in liquid nitrogen and stored at -20 °C unless stated otherwise.
4.2.15 Cultivation of E. coli for production of 6xHis-TF-AbWSD1 selenoprotein
Production of the 6xHis-TF-AbWSD1 selenoprotein was done according to Ausubel et al. (2001) and performed by Dr. Karin Kühnel with assistance of Felix Lambrecht. For the production of 6xHis-TF-AbWSD1 carrying selenomethionine instead of normal methionine, cultures were grown in a special M9 minimal medium supplemented with the appropriate antibiotics.
For expression, 250 ml of culture were incubated in a 1 l baffled flask in order to ensure good aeration of the culture. Flask and medium were equilibrated to 37 °C before inoculation. For inoculation of the medium, 1 ml of an overnight culture, grown in LB-medium, was pelleted, resuspended in M9-medium and used for inoculation of 250 ml M9 medium. Culture was incubated shaking at 180 rpm and 37 °C until it reached an OD600 of 0.3. Solid amino-acid supplements were added to the cultures to the final concentrations:
amount component 100mg/l L-Lysine 100mg/l L-Phenylalanine 100mg/l L-Threonine 50mg/l L-Isoleucine 50mg/l L-Leucine 50mg/l L-Valine
50mg/l L-Selenomethionine
METHODS
4.2.16 Cultivation of S. cerevisiae
Cultivation of S. cerevisiae expression cultures was done in accordance to Heilmann et al. (2012). In detail, 20 ml of SD-URA-media + 2 % galactose was inoculated to an OD600 = 0.05 from a preculture.
Precultures were grown in SD-URA + 2 % galactose at 30 °C and 180 rpm for 48 h. Expression cultures were incubated for five days at 30 °C and 180 rpm.