Single colony were isolated from petri dishes and resuspended in 20 l of ddH2O. The PCR reaction master mix comprised 400 nM of T7for (5 GCGAA ATTAA TACGA CTCAC TATAG G 3) and T7rev (5 GCTAG TTATT GCTCA GCGGT GGC 3), 200 M dNTP, 1 U Taq polymerase and 2 l of resuspended cells. The PCR program used was 94 °C 2 min, 94 °C 45 sec, 57 °C 30 sec, 72 °C 1 min/kb for 25 cycles finishing with additional double-length elongation step at 72 °C. This protocol was modified in a gene-specific manner by varying primers and Tm. After completion of the amplification reaction, it was mixed with 3 l loading blue and run on 1% agarose gel electrophoresis at 5-10 V/cm. Finally, the agarose gel was visualised at 360 nm UV with agarose gel illuminator.
Three different DNA sequencing services have been used throughout my work. Initially, EMBL-Heidelberg, Germany, then Macrogen, Korea and Cogenics, Grenoble were used.
Usually, about 5 l of 100 ng/l plasmid was sent for sequencing along with 20 l of custom primers at 5 pM. Usually, readouts were within 600-800 bp after the sequencing primer.
A single colony was grown in 5 ml overnight TB culture with the appropriate antibiotics.
Next, 50 ml TB supplemented with antibiotics was inoculated with a 1/200 dilution of the overnight and grown at 37 °C until the cell density reached was OD600 0.8. The culture was put immediately on ice and centrifuged in a refrigerated centrifuge (5804R, Eppendorf) at 4,000 g for 10 min. The supernatant was removed and pellet gently resuspended with 1 pellet volume of cold water, previously cooled on ice. The centrifugation step was repeated and supernatant removed. Then, the pellet was washed with 0.5 volumes cold water. The final
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wash was with 1/50 volume cold water and cells were either used directly for transformation or frozen with 10% glycerol at -80 °C.
Heat shock was performed usually with te cloning strain like Mach1 (Invitrogen). 12-50 l of cells were thawed on ice and incubated with 1 l ligation product for 1 min on ice then put at 42 °C for 42 s and immediately back on ice for 1 min. Recovery was performed with 200-1000 l SOC for 40-60 min at 37 °C in a shaking incubator, normally 180 rpm. For cloning, 50 l of transformant mix was streaked on LB agar petri dishes with the appropriate antibiotics and grown overnight at 37 °C.
Electroporation was performed with lab-prepared electrocompetent arabinose or IPTG inducibile BL21 (DE3) codon plus (RIL) cells. They were thawed on ice and 50 l were mixed with 10-100 ng plasmid in 10 mM Tris HCl pH 8. The mixture was transferred to a electroporation cuvette, also on ice. The electroporator (BioRad) was set to 1.8 kV and the cuvette subjected to an electric pulse. When an efficiency above 4.5 ms was achieved, cells were recovered in SOC medium and plated as described above.
Typically, 40 ml of resuspended culture was lysed in a French Press (FA-078, SLM Aminco) at 4 °C with pressure of 1000 psi and 3-4 cycles of the lysate through the cell (FA-072, SLM Aminco).
Sonication was performed at level 8 of the sonicator (XL2020, Misonix) for 4 min with total impulse alternating 10 s on and 30 s off. The lyses were performed on ice.
More recently for cell lysis, a Microfluidiser (M-110L, Microfluidics) was used at 18 kpsi, 4
°C. The lysate was cycled 10 times to assure good lysis.
A fresh starter culture of 5-10 ml was started from a single colony, 180 rpm, 18-20 h, 37 °C.
Typically, 50 ml - 1 L TB media supplemented with the appropriate antibiotics was inoculated with 1/100 dilution of the starter culture and grown at 37 °C, 180 rpm until OD600
0.6. Cultures were induced with 0.1 mM IPTG or 0.2% w/v arabinose depending on the promoter system of the strain. Usually, protein expression was carried out at 25 °C and 160 rpm overnight. Pellets were harvested at 8,000 rpm for 10 min with Eppendorf 5804R centrifuge or 6,000 g for 20 min in a large rotor centrifuge (Beckman). Pellets were resuspended in a ratio 1:1 to 1:2 with sonication buffer (50 mM Tris HCl pH 7, 300 mM NaCl, 5 mM imidazole pH 7, 5 mM ME, 1 tablet/50 ml Roche Complete Inhibitor and 1/1000 dilution of Benzonase). Sonication for 4 min alternating 10 s pulse and 30 s rest on ice was used for lysis. Total fractions were collected and soluble protein fractionated by centrifugation at 15,550g for 30 min. The resin for affinity purification was prepared for immobilisation of the target protein by washing with water and lysis buffer. Soluble fractions were passed through the resins in gravity columns and washed with 20 resin volumes (50 mM
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Tris HCl pH7, 300 mM NaCl, 5 mM imidazole and 5 mM -ME). A second wash of 10 resin volumes with 10 mM imidazole was performed and then elution either with buffer supplemented with 300 mM imidazole, or with increasing imidazole concentration steps of two resin volumes each: 100 mM, 200 mM, 300 mM and 400 mM.
Regularly, Bradford reagent (Sigma) was used for total and soluble fractions as well as purified fractions following protein expression tests. In such cases, either 990 l of Bradford reagent and 10 l of protein sample or volumes scaled twofold down were used against a calibration curve in the linear concentration range from 200 g/ml to 6mg/ml. These measurements were taken with a Biophotometer (Eppendorf) with calibration based on BSA.
When precise measurements were required, a ND-1000 spectrometer (NanoDrop) was used for measuring absorption at 280 nm of the sample diluted in an equal volume of 6 M GnHCl and concentration calculated using the computed extinction coefficient of the protein.
Usually, 0.75 mm thick gels 15% SDS-PAGE were used. The resolving gel consists of 5 ml 30% w/v acrylamide/bisacrylamide (Sigma), 2.5 ml Tris pH 8.8, 2.2 ml ddH2O, 50 l of 20%
w/v SDS solution, 7 l of TEMED (MP) and 50 l of 50% v/v ammonium persulphate solution. It was cast and overlaid with 100% EtOH. Then, the stacking gel mixture comprising 1.33 ml 30% w/v acrylamide/bisacrylamide, 2.5 ml Tris pH 6.8, 6.2 ml ddH2O, 50 l of 20% SDS solution, 7 l of TEMED and 50 l of APS. In case of high-throughput gels run on the the Ruby System (Amersham), gels were prepared by multiplying these volumes 3-fold. Occasionally, the large gels were supplemented with Rhinohide (Invitrogen) to increase their strength.
Transfer was done on either PVDF (Millipore) or nitrocellulose membranes (Amersham) for 1 h with constant 180 V in the BioRad dry blotter or Ruby western blotting module. When PVDF membranes were used activation for 15 s with 100% methanol preceded transfer with 1× Tris-Glycine and 10% Methanol. When nitrocellulose was used, the transfer was done with 1× Tris-Glycine and 20% ethanol. After completion of the transfer the membrane was washed with water and then 0.1% Tween-PBS for 5-10 min. Then, the membrane was usually blocked overnight with SuperBlock (37516, Thermo Scientific Pierce).
Initially, the proteins of interest along with a well-characterised protein as a control were expressed supplementing 50 M biotin in the culture medium. The control protein was purified to homogeneity and its concentration estimated by its absorbance at 280 nm. The control, well-characterised protein was used to prepare a calibration curve of known concentrations ranging usually from 0 to 2000 ng and then, both the proteins of interest and the calibration curve were transferred together to minimise transfer-related variation. Then, the protein blots were hybridised with streptavidin- Alexa488 (Amersham). Finally, they
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were visualised with a Typhoon fluorescence scanner (Amersham) and the digital images evaluated with ImageQuant (Amersham). The pixel intensities for each of the bands were measured and the calibration curve was prepared (Fig. 1). The latter was used for the estimation of the mass of protein visible on the western blot. Finally, the concentration of the protein was calculated taking into account the mass loaded.
FIGURE A.1: TYPICAL CALIBRATION CURVE USED FOR FLUORESCENT WESTERN BLOT QUANTIFICATION