5 Materials and Methods
5.2.1 Plasmid DNA purification
For preparative purposes a single bacterial colony was picked from an LB plate and used to inoculate 6 ml of LB medium containing the appropriate antibiotic. Cultures were allowed to grow overnight at 37 °C with vigorous shaking. Bacteria were harvested by centrifugation (14000 rpm, 1 min) and plasmid DNA was isolated by alkaline lysis and binding to anion-exchange matrices using the NucleoSpin® Plasmid (Macherey-Nagel), or GenElute Plasmid Miniprep Kit (Sigma-Aldrich) (see 5.1.2) according to the manufacturer’s instructions.
For preparative purposes and to yield larger amounts of plasmid DNA for transfection of mammalian cells overnight cultures of 100-400 ml were used.
Materials and Methods
71 Bacteria were harvested at 4 °C (4000 rpm, 30min) and plasmid DNA preparation was carried out using the NucleoBond® PC 100 Midiprep Kit (see 5.1.2). DNA concentration was determined by measuring absorption at 260 nm using the NanoDrop spectrophotometer (Witec AG).
For storage, 830 μl of bacterial cultures were mixed with 130 μl Glycerol (87 %), frozen in liquid nitrogen and stored at -80 °C.
5.2.2 Cloning
5.2.2.1 PCR (Polymerase chain reaction)
The polymerase chain reaction (PCR) serves as an in vitro-amplification system of DNA molecules (Mullis and Faloona, 1987). In order to generate the different constructs for protein expression in yeast as well as in mammalian cells either HA-UBA6 or pcDNA3.1-HA-FAT10 was used as a template in a PCR reaction together with the oligonucleotides indicated above (5.1.9.1) to amplify the desired DNA fragments. PCR conditions were optimized for the different fragments to yield enough PCR products for subsequent cloning steps. The different reaction mixtures with a total volume of 50 μl and the amplification conditions that were used are listed below:
UBA6
Components
Expand long template PCR system (Roche) Volume [ μl ]
10 x buffer 5
DMSO 2,5
dNTP-mix (10 mM each) 2,5
AA-113 Oligonucleotide forward (10 μM) 2 AA-114 Oligonucleotide reverse (10 μM) 2
pcDNA3.1-HA-UBA6 (350 ng µl-1) 1
DNA-Polymerase (Expand Long template) 1
H2O 34
72 PCR program: Touchdown PCR
Process Temperature [° C] Duration [sec] Repetition
Initial denaturation 94 240 1 x
Denaturation 94 60
Annealing 65 60
Elongation 68 210
15 x Δ 1°C
Denaturation 94 60
Annealing 50 60
Elongation 68 210
20 x
Final Elongation 68 600 1
Due to the size of the uba6 gene (3159 bp), it was not possible to obtain an amplification product with the correct size using standard PCR methods. Touchdown PCR uses a cycling program with varying annealing temperatures and thereby enhances the specificity of the initial primer–template duplex formation and hence the specificity of the final PCR product.
We chose an initial annealing temperature of 65 °C and in subsequent cycles (15 x), the annealing temperature is decreased in steps of 1 °C/cycle. Afterwards, a standard PCR program was applied for further 20x cycles with a final elongation step of 10 min at 68 °C.
FAT10
Components
Expand High Fidelity Plus (Roche) Volume [ μl ]
5 x buffer 2 (MgCl2) 10
DMSO 2,5
dNTP-mix (10 mM each) 2,5
AA-115: Oligonucleotide forward (10 μM) 2 AA-116: Oligonucleotide reverse (10 μM) 2
pcDNA3.1-HA-FAT10 (400 ng µl-1) 1
DNA-Polymerase (Expand High Fidelity Plus ) 1
H2O 29
Materials and Methods
73 PCR program:
Process Temperature [° C] Duration [s] Repetition
Initial denaturation 94 180 1 x
Denaturation 94 60
Annealing 60 30
Elongation 72 30
30
Final Elongation 72 480 1 x
5.2.2.2 Site-directed mutagenesis
All plasmids containing uba6 of fat10 that had been checked through sequencing contained at least one mutation in their sequence. To remove these mutations QuikChange Site-Directed Mutagenesis Kit (Stratagene) was applied. Primers (Microsynth) were designed that flanked the site of mutation with 10-15 nt overhangs on either side and used in a PCR reaction according to the manufacturer's instructions together with the mutated constructs as templates. Amplification was carried out with 18 cycles of melting (95°C for 30 s), annealing (Tm - 5°C for 60 s) and amplification (68°C for 60 s per 1 kb of DNA). PCR products were subsequently digested with 10 U DpnI for 1-3 h, heat inactivated repaired constructs were transformed into the E. coli strain XL10 Gold and the removal of mutations was verified by sequencing.
5.2.2.3 Agarose gel electrophoresis
After digestion of DNA with a restriction enzyme or purification of DNA, agarose gel electrophoresis was performed for both preparative and analytical purposes, using gels with an agarose concentration of 0,8-1,5 % and 0,5 μg ml-1 ethidium bromide in TAE buffer.
Samples were mixed with 6x loading buffer (Table 5) and and the mixtures transferred into the slots of the gel. The GeneRuler 1 kb DNA Ladder (see 5.1.1) was used as a DNA marker. For electrophoretic separation 100 V were applied. DNA was visualized by UV and PCR products were extracted and purified from agarose gels using the GenElute Gel Extraction Kit (Sigma-Aldrich) (see 5.1.2).
5.2.2.4 Restriction digest
The ability to cleave DNA at specific sites is one of the cornerstones of today’s methods of DNA manipulation. Restriction endonucleases are bacterial enzymes that cleave duplex DNA at specific target sequences with the production of defined fragments.
74 Site specific restriction endonucleases in the appropriate buffers (all Fermentas) were used to generate compatible ends that allow for ligation of DNA fragments into the respective restriction sites of the different vectors. The restriction sites had been included into the primer sequence and DNA fragments as well as the target vectors were digested with the same pairs of restriction enzymes. Digests were carried out using a standard protocol.
A typical digest mixture with a final volume of 100 μl contained 5 μg of plasmid DNA or respectively at least 1 μg of PCR product, 10 μl of 10 x restriction buffer and 1-10 U of enzyme. Mixtures were incubated at 37 °C for 2-5 h, according to the enzyme. Prior to ligation, the vector DNA was dephosphorylated to prevent self- or re-ligation for 30 min at 37 °C with the Calf Intestine Alkaline Phosphatase (CIAP), see 5.1.6. Dephosphorylation increases the effective concentration of available ends and efficiency of productive ligations.
The reaction was stopped by exposing the enzyme to 85 °C for 20 min.
Digested DNA and dephosphorylated DNA was subjected to agarose gel electrophoresis, and bands were visualized by ethidium bromide staining, cut out and subsequently purified from the gel slice using GenElute PCR Clean-Up Kit (Sigma-Aldrich) (see 5.1.2).
5.2.2.5 Ligation
Digested DNA fragments were ligated into designated vectors using 1 μl of the T4-DNA ligase and 2 μl of the matching 10x ligase buffer (Fermentas) in a total reaction volume of 20 μl. A 1:5 molar ratio of vector to insert was used whereas either 50 or 100 μg of vector were applied. Ligations were carried out in a thermocycler at 16 °C for 20 h. Afterwards, the T4 ligase was inactivated at 65 °C for 10 min.
5.2.2.6 Preparation of chemically competent E. coli cells
A single colony of bacteria (E.coli TOP10 F') was picked from an LB plate and used to inoculate 5 ml LB medium. Bacteria had time to grow overnight in an incubator at 37 °C and 200 rpm. Overnight culture was diluted at least 1/100 to inoculate 100ml LB medium. The bacterial culture was allowed to grow further to an OD600 of 0.5. The culture was cooled down on ice for 30 min before it was harvested through centrifugation at 4 °C with 4000 x g in a Sorval centrifuge for 20 min. Pelleted bacteria were carefully resuspended on ice in sterile 100 mM CaCl2, pelleted again (2200 x g for 30 min, 4 °C) and resuspended in 10 ml of a chilled 10 % glycerol-solution in CaCl2. Aliquots of 100 μl were filled in pre-chilled eppendorf tubes and frozen in liquid nitrogen before use or stored at -80 °C.
Materials and Methods
75 5.2.2.7 Transformation into E. coli TOP 10 F’
The ligations were transformed into chemically competent E.coli strain TOP10 F'. 100 μl of competent bacteria was thawed on ice and carefully mixed with 10 μl of the ligation mixture and incubated on ice for another 30 min to allow the plasmids to attach to the bacterial surface. Cells were then heat shocked for 40 s at 42 °C in a waterbath and transferred back on ice for 2 min. 400 μl of pre-warmed SOC medium without antibiotic was added and transformed bacteria were shaken for 1 h at 37 °C to allow development of antibiotic resistance, before they were streaked out on LB plates containing the respective antibiotic to select for successfully transformed bacteria.
5.2.3 Expression and purification of recombinant GST-TRIM11 from E. coli To purify recombinant GST-TRIM11 from E. coli BL21(DE3), 100ml HSG (Table 23) supplemented with Ampicillin (100 μg ml-1) was inoculated with a single colony of bacteria (E. coli BL21(DE3) GST-TRIM11) from LB agar plates including ampicillin and shaken at 37°C overnight. The overnight culture was diluted to a final OD600 of 0,1 in a total volume of 800 ml HSG and grown to exponential phase (OD600 2~3) followed by induction of the lac operon with 0,1 mM IPTG for 4 hours at 20 °C. Cells were harvested in a Sorvall RC6 Plus centrifuge (Thermo), at 18.000 rpm and 4 °C for 15min and pellets were resuspended in lysis buffer (Table 15) containing the protease inhibitors 0,1 mM phenylmethylsulfonyl fluoride (PMSF), 2 μg ml-1 aprotinin, 2 μg ml-1 leupeptin and 2 μg ml-1 pepstatin and disrupted 2 times in French press (2,5 kBar). Cell debris was removed by centrifugation at 18.000 rpm for 30 min at 4 °C. The supernatant containing the GST fusion protein was filtered through a 0,2 μm Filter and loaded onto a 1 ml Glutathione-SepharoseTM 4B column (GE Healthcare), with a flow rate of 1 ml min-1 using AKTAexplorer purification systems from GE Healthcare, as recommended by the supplier. 30 column volumes of PBS and GST high-salt washing buffer (Table 16) were used to wash unspecifically bound proteins off the column. GST-TRIM11 was eluted with 5 column volumes of GST-elution buffer (Table 19) containing 10 mM reduced Glutathione and subsequently dialyzed against PBS, concentrated and stored at 4 °C.