El cáncer y la Inanición de la Vida

2.9.1 DNA Extraction, Storage and Sequencing

All plasmid extractions from E. coli strains were performed using the Macherey Nagel Plasmid extraction kit and recommend steps according to producer’s manual instructions. For extraction

E. coli BL21 (DE3) lacIQ1 cells containing corresponding plasmids were grown in 3 mL LB media supplemented with corresponding antibiotics as overnight culture (250 rpm, 16 h, 37°C). Extracted and purified plasmids were quantified using a NanoDrop spectrophotometer prior to long time storage at -20°C. For sequencing 1 µg of plasmid DNA was sent for sequencing. Primers for sequencing can be found in appendix section of this thesis (Table 27). Obtained sequencing files were analyzed using CloneManager 9 software for instance by annealing the sequence data of variants with the template DNA sequence used for mutagenesis (see appendix Figure 89).

2.9.2 Polymerase Chain Reaction (PCR)

In vitro amplification of DNA was achieved by polymerase chain reaction (PCR) using Taq DNA or

high fidelity Phusion DNA polymerase.[113, 220] Reaction mixtures (if not stated otherwise) contained in a final volume of 50 µL: 1x DNA Polymerase Puffer; 5 U DNA Polymerase (Taq or Phusion); 0.2 mM dNTP mix; 20 ng plasmid DNA; 0.2 µM forward und reverse primer. Amplification of DNA was achieved in a thermocycler employing the program shown in Table 4.

Table 4. Thermocycler program for PCR amplification with Taq DNA polymerase.

Step Temperature [°C] Duration [sec]

Denaturation 94 20

25 cycles

Annealing 55 30

Elongation 72 60 per kb DNA#

Final Elongation 72 300

Storage 4 ∞

#

Elongation with Phusion DNA Polymerase was performed with 2 kb min-1 2.9.3 Oligonucleotide Design for PCR Amplifications

Primers were designed as recommended for efficient amplification of DNA fragments (GC content: 40-60 %; Tm: > 55°C; avoiding primer self- or hetero-dimers and hairpins). Designed oligonucleotides were re-checked with OligoAnalyzer Software from IDT (http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/).[221]

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2.9.4 Colony PCR

Colony PCR was performed to control correct assembly of generated DNA constructs. Therefore, a cell material from a colony grown on solid LB agar or 5 µL liquid cell culture were re-suspended in 50 µL ddH2O. Plasmid DNA was released by heating the sample to 95°C for 15 min. Cell debris was removed by centrifugation (20000 x g, 5 min, 20°C) and 5 µL from the supernatant was used in total 50 µL PCR mixture (section 2.9.2). All colony PCRs were performed using Taq DNA polymerase and the respective PCR amplification protocol (Table 4).

2.9.5 Phosphorothioate-Based Ligase-Independent Gene Cloning (PLICing)

In this thesis all DNA cloning steps were performed employing the phosphorothioate-based ligase- independent DNA cloning method (PLICing).[213] PLICing allows a nearly sequence independent cloning of DNA fragments, with high efficiency and very low background of false positive colonies. Making use of a chemical cleavage step to generate specific DNA overhangs for later hybridization, limitations of the widely used restriction-ligation cloning are circumvented. Figure 16 illustrates the principle of the PLICing cloning method (B) and the modification in the phosphorothiolated nucleotides applied in the protocol (A).

Figure 16. A schematic representation of the PLICing method (taken from Blanusa et al.)[213] A: Difference between natural phosphodiester bonds (upper) and the PTO modified nucleotide bonds (lower) employed in the PLICing method. B: Three step PLICing protocol for cloning of DNA fragments. The Figure was taken from Blanusa et al.[213]

In the first step, the amplification of insert and vector was achieved with modified (phosphorothiolated) primers employing a standard PCR protocol (section 2.9.2). The products were analyzed by agarose gel electrophoresis for quality of the amplified DNA (section 2.9.6). After DpnI digest and purification with a DNA purification kit, the PCR products were diluted to 0.03 pmol µL-1 insert and 0.01 pmol µL-1 vector, respectively. Cleavage of phosphorothiolated nucleotides was achieved by mixing 4 µL of insert DNA and 4 µL vector DNA, each with 2 µL of iodine cleavage mix

38 (6 mM I2 in EtOH; 250 mM Tris-HCl pH 9.0). The samples were heated in a thermocycler (5 min, 70°C) for cleavage of PTO nucleotides from 5’endings. The mechanism for cleavage of PTO containing DNA was elucidated by Eckstein and Gish[222] and is shown exemplary in Figure 17.

Figure 17. Chemical cleavage of phosphothiolated nucleotides in the presence of iodine and ethanol (I2/EtOH)

under alkaline conditions. In step one the sulfur atom is alkylated by iodoethanol leading to an instable intermediate which releases the phosphorothiolated nucleotide from the DNA. The detailed mechanism was introduced by Eckstein and Gish in 1989.[222]

Cleaved DNA fragments were mixed and transformed by heat shock into chemical competent E. coli cells (section 2.8.2) without further purification step.[124, 213, 223] The obtained clones were analyzed by colony PCR and agarose gel electrophoresis (section 2.9.4 and 2.9.6). One colony containing the construct in expected size was used for plasmid extraction and preparation of glycerol stocks. Correct assembly of the plasmid was verified by sequencing of the generated DNA constructs.

2.9.6 Agarose Gel Electrophoresis and DNA Quantification

Quality control of all DNA samples (PCR products, enzymatic restriction, plasmid DNA) was achieved by gel electrophoresis on 1 % agarose gels. Smaller DNA fragments (<500 bps) that were generated during development of OmniChange[223] (3.7) and PTRec[124] (3.8) were analyzed on 2 % agarose gels for higher resolution. Solid agarose powder was solubilized by heating in Tris EDTA acetate buffer (TAE-buffer; 40mM Tris; 2 mM EDTA; pH 8.0) in a kitchen microwave. Five µL of DNA samples were mixed with 6x loading dye. Samples were loaded on an agarose gel and separated for 30 min at 110 mV (2 h for 2 % agarose gels). Staining of DNA was achieved employing ethidium bromide (40 μL ethidium bromide solubilized in 1000 mL ddH2O). Visualization of DNA samples on agarose gels was achieved on a UV illuminator connected with a gel scanning system. Quantification of DNA (plasmid DNA or PCR products) was done by using a NanoDrop spectrophotometer.

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2.9.7 Cloning of P450 BM3 Wild-Type into pALXtreme-1a Expression Vector

Smaller plasmids have significant advantages in directed evolution campaigns due to a higher efficiency during amplification of DNA as well as increased transformation efficiencies.[224] Therefore the P450 BM3 WT gene was cloned from pET-28a(+) into pALXtreme-1a using the PLICing protocol[213] which leads to a decrease in plasmid size of more than 30 % (section 2.6). The primer sequences employed during cloning can be found in the appendix section of this thesis (Table 27). The generated construct was sequenced and used for all engineering work and characterizations of P450 BM3 variants (see full DNA sequence of P450 BM3 WT in appendix section).

2.9.8 Site Directed and Site Saturation Mutagenesis

Focused mutagenesis is a standard approach in protein engineering for the substitution of single amino acids in protein sequences in a defined manner.[32, 36, 98, 103] The most common protocol for site directed mutagenesis (SDM) and site saturation mutagenesis (SSM) is the whole plasmid amplification using complementary synthetic oligonucleotides also known as the “QuikChange protocol”.[126, 225] Main challenge of this protocol are the complementary primers which prevent efficient DNA amplification due to formation of strong heterodimeric structures which is exemplary shown for saturation of positions R47 and Y51 (P450 BM3) in this thesis (Figure 18).[223]

Figure 18. Simultaneous site saturation mutagenesis of rationally selected amino acid residues R47 and Y51 of P450 BM3. The principle for hybridization of complementary primers containing NNK-degenerated nucleotides to a circular plasmid is shown. PCR amplification is in 3’-direction, catalyzed by Phusion DNA Polymerase.

In this thesis a modified two-step PCR protocol is used to obtain higher yields of PCR products during generation of focused mutant libraries by reducing formation of heterodimers of oligonucleotides.[221] All primers were designed according to recommendations provided in the QuikChange SDM manual and using OligoAnalyzer Software from IDT (www.eu.idtdna.com/analyzer/applications/oligoanalyzer).[125, 226] In step 1 of the modified protocol the PCR is performed with single primers for three cycles (section 2.9.2) to generate linear

templates. Step 2 comprises mixing of both reaction mixtures from step 1 and performing 15 additional PCR cycles under identical conditions. After 15 cycles of exponential DNA

amplification, the samples were analyzed for PCR product quantity and quality by agarose gel electrophoresis (section 2.9.6). Figure 79 in appendix section displays PCR products from SSM at position F87 of P450 BM3. Correctly amplified products were supplemented with 10 U of DpnI

40 restriction enzyme to remove methylated template DNA (16 h at 37°C). Digested PCR products (5 µL) were directly transformed into chemical competent E. coli lacIQ1 cells (section 2.8.2). Obtained colonies were used to prepare mutant libraries for screening in MTP format (section 2.8.6).