Materials and methods: biochemistry

General operations

Sterilized equipment and laboratory glassware was used in all biochemical experiments described here. Single-use items were collected and autoclaved after usage and prior to disposal. Reusable materials were autoclaved after usage and subsequently sealed with sterile aluminum foil. All experiments, which involved the work with genetically modified organisms, were performed under a laminar flow biological safety cabinet Bio-II-A from Prettl-Telstar. Aqueous media and solutions were prepared with sterilized MilliQ water.

Materials

Luria-Bertani (LB)-media: Tryptone (1%, w/v), yeast (0.5%, w/v) and NaCl (1%, w/v) were dissolved in MilliQ water and autoclaved at 120 °C for 25 min.

LB plates: Tryptone (1%, w/v), yeast (0.5%, w/v), NaCl (1%, w/v) and agar (7.5%, w/v) were dissolved in MilliQ water and autoclaved at 120 °C for 25 min. After cooling to 50 °C, carbenicillin was added to the LB-media, which was subsequently casted into sterile Petri dishes.

Antibiotics: Carbenicillin (100 g/mL) was used as additive in the prepared media to suppress undesired bacterial growth except for the resistant E. coli host strain ER2566.

Bacterial host strain: The commercially available E. coli host strain ER2566 (New England Biolabs) was used for protein expression, which was made competent prior to use.

Preparation of competent E.coli cells

The preparation of competent E. coli cells facilitates the uptake of plasmid DNA through the bacterial cell membrane. Thus, a single colony was picked from a LB plate and transferred into 5 mL of LB medium. The cultivation was performed at 37 °C overnight before 2 mL of the medium was inoculated into an Erlenmeyer flask, which contained another 50 mL of LB medium. The medium was cultivated at 37 °C until an OD600 of approximately 0.3 was obtained. The cell culture was immediately cooled on ice and was subsequently centrifuged at 3.000 rpm and 4 °C for 15 min. The supernatant liquid was removed and an aqueous solution of CaCl2 (0.1 M, 10 mL, 5 °C) was added to the cell pellet. The cell suspension was incubated on ice for 20 min followed by centrifugation at 3.000 rpm and 4 °C for 15 min. After the removal of the liquid components, an aqueous solution of CaCl2 (0.1 M, 5 mL), which also contained glycerol (15%) was added to the cell pallet. The solution was divided into sterile

Eppendorf safe-lock tubes (500 L each) that were frozen in liquid nitrogen and stored at –80 °C.

Vectors: The commercially available (New England Biolabs) plasmid vector pTXB1 was used for the recombinant protein expression of the Zf12 domain. The vector also included a gene for carbenicillin resistance.

Transformation of competent E. coli cells

A 500 L glycerol stock of competent E. coli cells was slowly thawed on ice. Subsequently, 5 L of the plasmid DNA was added to 100 L of the E. Coli stock and the sample was incubated on ice for 30 min. The sample was heat-shocked at 42 °C for 45 sec and placed on ice again for 5 min. LB medium (900 L) was added and the sample was incubated at 37 °C for 1 h. The solution was centrifuged at 3.000 rpm for 2 min. 100 L of the supernatant was placed on a LB plate, which was subsequently incubated at 37 °C overnight.

Recombinant protein expression

A single colony was picked and transferred to LB medium (5 mL), which also contained 5 L of carbenicillin. The medium was cultivated at 37 °C for 24 h. Afterwards, the medium was used to inoculate the expression culture that was cultivated in 400 mL of LB medium containing an appropriate amount of carbenicillin. The expression culture was incubated at 37 °C for 4 h before 40 L of IPTG (1 mM) was added and the incubation was continued at 16 °C for 24 h. The suspension was centrifuged at 4.000 rpm and 4 °C for 20 min. The supernatant was removed and the cell pallet was transferred into a centrifuge tube before the cell lysis reagent BPer (1 g/mL) was added. The suspension was placed on ice and was subject to pulsed sonification (5 x 45 sec, 50% power, 50% impulse) using a Sonoplus GM 7 (Bandelin). The cell material was separated by centrifugation (14.000 rpm, 4 °C, 20 min) and the supernatant that contained all expressed proteins was purified by affinity chromatography.

Purification of Zf12

The cell lysate was purified by affinity chromatography using Chitin beads (New England Biolabs). Hence, the expressed Zf12 domain contained a fused chitin binding domain (CBD), which showed high affinity for the corresponding chitin beads. To do so, the chitin beads (100 mL) were loaded to a XK26/20 column (GE Healthcare) and were equilibrated with two bed volumes of water and ten bed volumes of column buffer (20 mM HEPES, 500 mM NaCl, 0.1% Tween20, 20 mM TCEP, pH 8) using an Äkta Prime Plus system (GE Healthcare). The cell lysate was repeatedly loaded onto the column at a flow rate of 0.5 mL/min overnight,

whereby only the Zf12 protein stuck to the chitin beads. All other proteins were washed from the column using 20 bed volumes of column buffer at a flow rate of 2 mL/min. The Zf12 thioester protein was liberated from the chitin beads by adding 1.5 bed volumes of cleavage buffer (20 mM HEPES, 500 mM NaCl, 0.1% Tween20, 20 mM TCEP, 250 mM MeSNa, pH 8) and incubation overnight. The desired protein was eluted by adding column buffer at a flow rate of 2 mL/min until the detected UV absorbance as well as the electric conductivity reached a minimum. The aqueous solution was immediately lyophilized and the solid components were solubilized again in the minimum amount of water required to obtain a homogeneous solution. Subsequently, the solution was subject to RP-HPLC purification, the peptide was lyophilized and stored at –20 °C.

Oligonucleotide annealing protocol

The DNA oligomers were purchased as a freeze-dried solid, and were therefore, dissolved in 900 L MilliQ water. The absorption values were determined by means of UV-vis spectroscopy and the concentrations were calculated using Lambert-Beers law with the molar absorption coefficients provided by the DNA supplier. Distinct concentrations of complementary DNA strands were pipetted into an Eppendorf safe-lock microcentrifuge tube, which contained the annealing buffer (10 mM Tris, 50 mM NaCl, pH 7.8). The tube was sealed and heated at 95 °C for 5 min and subsequently cooled to room temperature. Prior to use, the samples were centrifuged at 6.000 rpm using a Roth microcentrifuge.

Gel electrophoresis

Polyacrylamide gel electrophoresis (PAGE) was used to examine the binding ability of the engineered zinc-fingers for their DNA target sequence as well as to test for their DNA hydrolysis capacity. Gel electrophoresis is used to separate different macromolecules according to their electrophoretic mobility, which is dependent on the length, conformation and intrinsic charge of the analytes.

Polyacrylamide gel electrophoresis (PAGE)

Polyacrylamide gels are cross-linked porous materials having small pore sizes, which are suitable for the separation of polynucleotides with a length between 5–500 bp. The gel matrix was prepared by co-polymerization of acrylamide with N,N′-methylenebisacrylamide in the presence of APS and TEMED. The Zf13 peptides as well as the DNA samples were pipetted into the wells of the polyacrylamide gel and electrophoresis was performed with a suitable running buffer and by applying an appropriate voltage. The electrophoretic migration of the macromolecules was evaluated by the detection of the fluorescent 6-FAM label that was attached to the 5′-end of a single DNA strand. This was conducted on a Typhoon 9400

Variable Mode Gel Imager from Amersham Biosciences (GE Healthcare) by scanning the gel with the blue laser channel and using ImageQuant software for visualization.

Native PAGE

Native PAGE is performed under non-denaturing conditions that allow the detection of higher-order structures, such as peptide/DNA complexes after electrophoresis. This method was used to evaluate the binding ability of the engineered zinc-fingers towards their consensus dsDNA sequence.

Native polyacrylamide gels were prepared according to the following procedure using the Mini-PROTEAN^® Tetra Handcast Systems (Bio-Rad). The glass plates and the spacer plates were washed with double-distilled water followed by an aqueous solution of EtOH (70%) and were allowed to dry in air. The plates were assembled to generate a gel thickness of 0.75 mm. The cassette was placed on a gasket and filled with a freshly prepared acrylamide gel solution until the solution was 1 cm below the teeth of the comb. A layer of distilled water was rapidly but carefully added on top of the acrylamide gel and the gel was allowed to polymerize. Subsequently, the water was removed using filter paper. The remaining space in the cassette was filled completely with stacking gel and a comb was immediately placed in the cassette. After complete polymerization, the cassette was transferred to the electrophoresis apparatus, which was filled with running buffer. After the addition of 2 L glycerol (30%) to each sample, the comb was removed and the samples were pipetted into the wells of the gel. Electrophoresis was performed by applying a constant voltage of 170 V.

All buffers and solvents were prepared under the exclusion of denaturing or metal-chelating substances, such as EDTA or SDS, to maintain native conditions.

10x TB Buffer (1 L): 108 g Tris 55 g Boric acid adjusted to pH 8.0

Gel Solution (50 mL): 15 mL/25 mL Rotiphorese Gel 40 for 12%/20% acrylamide gel 5 mL of 10x TB buffer


270 L of aqueous APS (10%) 27 L of TEMED

Final volume adjusted to 50 mL with MilliQ water

SDS-denaturing PAGE

Polyacrylamide gel electrophoresis under denaturing conditions was performed to evaluate the DNA cleavage ability of the engineered zinc-finger mutants towards their consensus DNA sequence. In contrast to native PAGE, denaturing PAGE eliminates all non-covalent interactions allowing for the detection of smaller and therefore faster migrating DNA fragments, which resulted from a possible hydrolysis. The polyacrylamide gels were prepared according to the procedure described above that however differs in the fact that the gel solution as well as the running buffer contained an additional 0.1% SDS. 2 L of TriTrack DNA loading dye (Fermentas) was added to the samples, which were subsequently loaded on the gel. Electrophoresis was performed by applying a constant voltage of 170 V.

6x TriTrack Loading Dye: 10 mM Tris HCl (pH 7.6) 60 mM EDTA

0.03% bromophenol blue 0.03% xylene cyanol FF 0.15% orange G

Glycerol (60%)