Work with E coli

4.2.1.1 Cultivation of bacterial cells

Bacterial strains were cultivated at 37°C in liquid LB medium containing antibiotic(s) (see Table 8) for plasmid selection. Culture growth was monitored by measuring the absorbance at 600 nm to determine the optical density of cell cultures (OD600).

Cultivation on solid agar plates was performed in plates containing LB medium and the required antibiotic(s) for plasmid selection. Individual colonies or aliquots of glycerol stocks were streaked out using sterile disposable inoculation loops. Plates were incubated upside down at 37°C for not longer than 24 hours. Short-term storage of the bacterial clones was possible at 4°C.

4.2.1.2 Transformation of electro-competent bacterial cells by electroporation

A 75 µl-aliquot of electro competent E. coli cells XL1 blue MRF was thawed on ice and 3 µl of a ligated plasmid sample (see 4.2.3.6 or 4.2.3.10) or 0.5 µl of a plasmid was added and mixed. The mixture was transferred to cold 0.2 cm cuvettes for electroporation and the pulse was performed in a Bio-Rad MicroPulser Electroporation apparatus using program EC2. After the electroporation, 1 ml of LB medium was added and the sample was incubated for 45 minutes at 37°C. The suspension was then transferred to a 1.5 ml-tube and centrifuged at 4,000 rpm for 4 minutes at RT. 900 µl of the supernatant was discarded. The pellet was resuspended in the remaining volume, plated on LB supplemented with antibiotic(s), and incubated overnight at 37°C. For re-transformed plasmids, centrifugation step was skipped and 100 µl of regenerated cells was directly plated and incubated overnight at 37°C.

4.2.1.3 Transformation of chemo-competent bacterial “Rosetta Star” cells by heat shock

A 75 µl-aliquot of chemo competent E. coli cells Rosetta Star was thawed on ice and 0.5 µl of an E. coli expression plasmid pExJPF (see Table 2) was added and mixed. The mixture was transferred to 1.5 ml-tube and incubated for 5 minutes on ice. The heat shock was performed for 45 seconds at 42°C. After a second incubation step for 5 minutes on ice, the cells were supplemented with 1 ml LB medium and incubated for 2 hours at 37°C. 100 µl of regenerated cells was plated on LB supplemented with antibiotics Kanamycin and Chloramphenicol and incubated overnight at 37°C.

4.2.1.4 Long-term storage of plasmid DNA in E. coli

Plasmids to be stored were transformed into electro-competent E. coli cells (see 4.2.1.2). Single colonies were streaked out as dense patches in the appropriate medium and

incubated overnight at 37°C. Cells were recovered with inoculation loops and suspended in 1 ml sterile 20% (v/v) glycerol. Glycerol stocks were stored at -80°C.

4.2.1.5 Purification of plasmid DNA from E. coli (“mini-preparation”)

Plasmid DNA was isolated from E. coli using the peqGOLD Plasmid Miniprep Kit II from Peqlab according to manufacturer’s instructions. Bacterial cells used for mini preparation were streaked out as dense patches on LB supplemented with antibiotic(s) and cultivated overnight at 37°C. Cells were collected by a sterile disposable inoculation loop and suspended in 250 µl “Solution I” (second step in manufacturer’s manual). Elution of DNA was performed with 100 µl H2O.

4.2.1.6 Heterologous expression of HIS-tagged yeast proteins in E. coli

For heterologous expression of yeast protein Pol5 or Pol5 domains in E. coli, one single colony of “Rosetta Star” cells containing the respective E. coli expression plasmid (see 4.2.1.3) was used to inoculate 100 ml LB+KAN+CHL liquid medium (see Table 8). The culture was incubated overnight at 37°C and the next day, it was used to inoculate 1 L LB+KAN+CHL liquid culture to an OD600 of 0.1. After growth at 37°C to an OD600 of about

0.8, the protein expression was induced by a concentration of 0.1 mM IPTG in the liquid culture and cells were incubated for 6 h at 24°C. Subsequently, cells were harvested by centrifugation at 5,000 rpm for 10 minutes at RT. Resulting cell pellet was resuspended in 40 ml of supernatant medium, transferred to 50 ml-falcon tube, and centrifuged again at 4,500 rpm for 3 minutes at 4°C. Pellets were frozen in liquid nitrogen and stored at -20°C.

4.2.1.7 Affinity purification of HIS-tagged yeast proteins expressed in E. coli

Cell pellet corresponding to 1 L E. coli culture, which was obtained as described in section 4.2.1.6, was thawed completely on ice and resuspended in 10 ml EC (P1P) Extraction Buffer (see Table 9) using a syringe and a cannula. After addition of 1.5 g Zirconia-Silicate beads, cell lysis was performed on a vortexer by 10 steps consisting 30 seconds vortex and 30 seconds cooling on ice. The lysate was centrifuged at 15,000x g for 15 minutes at 4°C and transferred to new 50 ml-falcon tube to separate the Zirconia-Silicate beads. The resuspension step with syringe and canula as well as the cell lysis with Zirconia-Silicate beads and vortex was repeated once more. Next, the lysate was centrifuged at 15,000x g for 5 minutes and 4°C and transferred to new 15 ml-falcon tube. Volume of the lysate was adjusted to 12 ml with EC (P1P) Extraction Buffer, supplemented with 1% Triton X-100 and 5 µg/ml RNase A, and incubated for 10 minutes at 4°C under gentle rotation. After that, the lysate was clarified by centrifugation at 15,000x g for 30 minutes at 4°C.

The clarified whole cell lysate (WCL) was transferred to a new 15 ml-falcon tube and 1% of the WCL was collected, mixed with 6x Laemmli Buffer, and stored at -20°C for subsequent SDS-PAGE analysis (see 4.2.5.2) by Coomassie staining (see 4.2.5.3) and western blotting (see 4.2.5.4). Protein concentration was determined using Bradford

Assay (Protein Assay “Dye Reagent Concentrate”, Bio-Rad) according to manufacturer’s indications (see 4.2.5.1). Rest of the WCL was added to a new 15 ml-falcon tube containing 200 µl slurry of Ni-NTA agarose beads. The beads had been previously equilibrated with EC (P1P) Extraction Buffer containing 1% Triton X-100. The WCL was incubated with beads for 2 hours at 4°C under gentle rotation.

Afterward, lysate suspension was centrifuged at 800 rpm for 3 minutes at 4°C and 1% of supernatant was collected, mixed with 6x Laemmli Buffer, boiled at 95°C, and stored at -20°C as flow-through (FT) for subsequent SDS-PAGE analyses. Remaining supernatant was discarded and first washing step was performed by adding 10 ml EC (P1P) Extraction Buffer, resuspending the beads, and centrifuging at 800 rpm for 1 minute at 4°C. Supernatant was discarded and beads were resuspended again in 10 ml EC (P1P) Extraction Buffer. Suspension was completely transferred to PolyPrep Chromatography Column. Beads were washed ten times with 10 ml EC (P1) Wash Buffer (see Table 9) each time. 1% of flow-through from the first, fifth, and last washing step (W1, W5, W10) was collected, mixed with 6x Laemmli Buffer, boiled at 95°C, and stored at -20°C for analyses. For protein elution, the first elution step was performed by adding 500 µl EC (P1) Elution Buffer (see Table 9) to column and eluate 1 was collected. For the second elution step, beads were resuspended in 1 ml EC (P1) Elution Buffer, the suspension was transferred to 1.5 ml-tube of low protein binding, which was incubated under rotation for 30 minutes at 4°C. Afterward, the elution fraction was collected from the supernatant after centrifugation at 800 rpm for 1 minute at 4°C and it was saved as eluate 2. Last elution step was performed in 1 ml EC (P1) Elution Buffer by incubating the bead suspension under rotation overnight at 4°C. Eluate 3 was obtained as described for eluate 2. 100 µl of each eluate was mixed with 6x Laemmli Buffer, boiled at 95°C, and stored at -20°C for subsequent SDS-PAGE analyses. Finally, beads were resuspended with 1 ml EC (P1) Elution Buffer and one 100 µl-aliquot of suspension was mixed with 6x Laemmli Buffer (Beads after Elution, BaE). Samples BaE were boiled for 10 minutes at 95°C and stored at -20°C for analysis. Rest of eluates and remaining beads were stored at 4°C.