Cloning methods

Restriction endonucleases were always used together with adequate buffers that were provided by the producer. If two restriction enzymes were used in the same reaction, possible overdigestion (also: star activity) had to be taken into account. Digestion with restriction enzymes occurred at different steps during the cloning process:

• Digestion with two adequate restriction enzymes to obtain the desired insert and vector cut at chosen restriction sites, so that they were prepared for subsequent ligation

• Digestion of recombinant plasmid with one adequate restriction enzyme prior to transformation of competent Pichia cells

• Digestion with DpnI for elimination of residual template DNA after PCR

Table 26 Pipetting scheme for restriction digestion prior to ligation

Reagent Volume [µL] Reaction conditions

Template 10

Incubation for 2 to 3 hours at 37°C

10 x buffer 5

Restriction enzyme I 1 Restriction enzyme II 1

UHQ-H2O 33

Total volume 50

Table 27 Pipetting scheme for restriction digestion prior to Pichia transformation

Reagent Volume [µL] Reaction conditions

Template (recombinant plasmid) 10 Incubation for 1 hour at 37°C

Deactivation of restriction enzymes at 80°C for 20

minutes

Restriction enzyme 0.5

10x buffer 1.5

UHQ-H2O 3

Total volume 15

Table 28 Pipetting scheme for restriction digestion with DpnI

Reagent Volume [µL] Reaction conditions

PCR product 30 Incubation for 1 to 2 hours at

37°C

Deactivation of restriction enzyme at 65°C for 20

minutes

10 x Tango buffer 3

DpnI 1

Total volume 34

4.2.2.2 Ligation

When cohesive ends, which are created by restriction endonucleases, associate, the joint still shows some nicks in both strands. These nicks are sealed in vitro with purified DNA ligase; the enzyme seals single-stranded nicks between adjacent nucleotides in a doubled-stranded DNA chain. (Primrose et al., 2010) Ligation was performed according to the “Rapid DNA Ligation Kit Protocol, Thermo Scientific”:

Table 29 Pipetting Scheme for Rapid DNA Ligation

Reagent Volume [µL] Reaction conditions

Linearized vector DNA 0.5 Incubation for 5 minutes at

room temperature

The ligation mixture was directly used for E.coli NEB5α

transformation (see below).

Insert DNA (Cgl1, PnGOx) 2 5x Rapid Ligation Buffer 4

T4 DNA Ligase 1

Water, nuclease-free 12.5

Total volume 20

4.2.2.3 Transformation

Competent strains were obtained from the culture collection of the Institute of Food Biotechnology. The choice of either electroporation or chemical transformation of a cloning strain simply depended on the current preference of higher transformation efficiency (electroporation) or a faster and more convenient process (chemical transformation).

4.2.2.3.1 Transformation of chemically competent E.coli cells (Cloning strain)

Chemical transformation of competent E.coli NEB5α cells (New England BioLab^®) was executed as suggested in the “High Efficiency Transformation Protocol (C2987H/C29871)” (New England BioLabs^®) with small changes: competent cells were thawed on ice for 10 minutes. For one transformation 5 µL of plasmid DNA were added to 50 µL of those cells, mixing was done carefully by flicking of the tube. After the mixture was stored on ice for 30 minutes, the cells were heat shocked at exactly 42°C for 1 minute and afterwards put on ice again for 5 minutes. 250 µL of SOC medium at room temperature were added and the cells were then incubated at 37°C and 125rpm. Finally, each 50 µL and the remaining volume of the cell mixture were spread onto pre- warmed selective plates and incubated upside down overnight at 37°C.

4.2.2.3.2 Transformation of electrocompetent E.coli cells (Cloning strain or expression strain) Electroporation was used either to transform a competent cloning strain, E.coli DH5α, or a competent expression strain, E.coli BL21*(DE3). The preparation of electroporation involved thawing electrocompetent cells on ice for about 10 minutes and UV light treatment of reused electroporation cuvettes before they were also put on ice. 1 µL of plasmid-solution was gently mixed with 50 µL cell suspension and incubated for 5 minutes on ice. Then, the cell suspension was put into the electroporation cuvette, the cuvette was put into the micropulser and the cells were treated with the pre-set program ‘Ec1’ of the micropulser. Afterwards the cells were resuspended with SOC medium, this mixture was transferred to an Eppendorf tube and incubated at 37°C and 125 rpm for 1 hour. Each 50 µL and approximately 100 µL of a 1:5 dilution of the incubated cell suspension were spread onto selective plates and incubated upside down overnight at 37°C.

4.2.2.3.3 Transformation of electrocompetent P. pastoris cells (Expression strain)

4 µL of linearized plasmid were added to 50 µL of electrocompetent Pichia pastoris X-33 cells.

This mixture was added to the side of a chilled cuvette, and the cuvette was then placed into the micropulser. The electroporation was carried out at a voltage of 1.5 kV and a pulse length of 3 msec. 500 µL ice cold 1 M sorbitol and later 500 µL YPD, pH 7.5 were used to resuspend the cells. The sample was incubated at 30°C and 120 rpm for 4 hours. 150 µL of the incubated cell suspension were spread onto selective plates. After centrifugation of the remaining cell suspension at 5000 rpm for 30 seconds 700 µL of supernatant were discarded, then the cells were resuspended in the remaining volume and 200 µL of this concentrated cell suspension were spread onto selective plates. Both plates were incubated upside down at 30°C.

4.2.2.4 Agarose gel electrophoresis

Applying an electric field to DNA fragments in supporting gel medium results in separation of those DNA fragments depending on their molecular weight. (Chrambach et al., 1987) Agarose gel electrophoresis is the standard method for separation, DNA restriction fragment-analysis and purification of DNA and RNA fragments. (Westermeier R., 2005) The 0.8% (w/v) agarose gel was prepared by melting 0.8 g Agarose in 100 mL 1x TAE – buffer in a microwave, by tempering the molten gel to 60°C and by subsequently pouring the molten gel into a gel cast. 2.7 µL of peqGreen DNA dye were added to the still liquid gel for later visualization of the bands by UV light and mixed properly. When cooled down, the gel was put into a tray filled with 1x TAE buffer in a way that the buffer totally covers the gel, then the samples were filled into the wells of the gel. The standard used to provide a reference for the molecular weight of DNA was Thermo Scientific GeneRuler™ DNA Ladder Mix (Figure 14). Finally, the gel was exposed to an electric field of 90 V until the visible blue loading dye line reached the middle of the gel and the DNA bands were visualized by UV light using the Molecular Imager^® Gel Doc™ XR+ (BIO-RAD). The desired bands were excised with a scalpel and the DNA was extracted using an illustra™ GFX™

PCR DNA and Gel Band Purification Kit (GE Healthcare).

Figure 14 GeneRuler DNA Ladder Mix, Thermo Scientific

4.2.2.5 Sequencing

DNA sequencing was performed to verify if the insertion of the ordered genes – of Cgl1 into pET-21a+ and of PnGOx into pPICZαA and pGAPZαA- was successful. Later, sequencing was also used to check if site-directed mutagenesis led to the desired addition of six bases to the PnGOx gene in pPICZαA. The samples for sequencing were prepared by mixing 7.5 µL DNA and 7.5 µL water with 3 µL of either forward or reverse primer, and then they were analyzed by Microsynth (Wolfurt, Austria). The sequencing results were examined using the multiple sequence alignment program Clustal Omega and the DNA sequence converting program Reverse Complement.

4.2.2.6 Site-directed mutagenesis – DpnI method for PCR mutagenesis of circular DNA For site-directed mutagenesis two primers were used: The 5’-primer served as mutagenic oligonucleotide, in other words it contained the desired mutation (here: 6 additional bases). A second primer, the 3’-primer, bound adjacent to the mutagenic primer on the opposite strand of the double-stranded plasmid. DNA polymerase (here: Phusion DNA polymerase) initiated synthesis of the DNA at each oligonucleotide; the final PCR product was a linear plasmid containing the desired mutation. (Brenner & Miller, 2002)

Table 30 Phusion PCR pipetting scheme for site-directed mutagenesis

Reagent Volume [µL] Reaction conditions

Phusion Mastermix 12.5 A program was written on the PCR

Thermocycler to create an annealing temperature gradient, so that annealing

reactions could happen at three different temperatures in a range from 62°C to 71°C (namely 62.6°C, 65.6°C,

67.7°C).

Template (pPICZαA-PnGOx) 1 Primer 5GOxmut1 (1:10 dilution) 1 Primer 3GOxmut2 (1:10 dilution) 1

UHQ-H2O 10.5

Total volume 26

Processing of the PCR product

• Subjection of PCR products to agarose gel electrophoresis

• Gel extraction according to Kit specifications

• Chemical transformation to competent E.coli NEB5α cells (linear DNA is transformed to circular DNA in E.coli cells)

• a DpnI restriction digest was performed to specifically cut the template DNA at methylated sites, and to preserve only the PCR product

• MiniPrep according to Kit specifications

• Linearization of mutated plasmid by restriction digest with SacI

• Transformation by electroporation into competent Pichia pastoris X-33 cells