Microbial Enzymes and Biotransformations Microbial Enzymes and ...

66 Brakmann and Stumpp
14. DNA sequencing kit (e.g., FS kit, Applied Biosystems, Foster City, CA) and
sequencer.
2.4. Identification and Characterization of Selected Variants
1. T7 RNA polymerase preparation of wild-type, as well as variant, enzyme (according
to [21]); final enzyme concentration 1–5 µg/µL.
2. 10X transcription buffer: 400 mM Tris-HCl, pH 8.0, 80 mM MgCl 2 , 100 mM NaCl,
20 mM spermidine, and 300 mM dithiothreitol.
3. Linear template with a T7 promotor, e.g., plasmid pACYC-T7Tet.
4. Oligonucleotide primers for sequence analysis of the transcribed gene.
5. Nucleoside triphosphates (100 mM each).
6. Phenol, equilibrated with TE buffer, pH 7.5–8.0.
7. TE buffer: 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA.
8. Chloroform, p.A.
9. 3.5 M sodium acetate.
10. Ethanol, p.A.
11. Agarose gel electrophoresis equipment.
12. Kit for the elution of nucleic acids from agarose gels, e.g., QIAquick Spin Kit
(Qiagen, Valencia, CA).
13. Reverse transcriptase and buffer.
14. Vent polymerase, exonuclease-deficient.
15. Pfu DNA polymerase.
16. 10X PCR buffer: 100 mM Tris-HCl, pH 8.5, 500 mM KCl, 0.01% gelatin, and 15
mM MgCl 2 .
17. Deoxynucleoside triphosphates (10 mM each).
18. TA cloning vector, e.g., pCR2.1 (Invitrogen).
19. Competent E. coli cloning strain, e.g., XL1-Blue (Stratagene).
20. DNA sequencing kit and sequencer.
3. Methods
The methods described below outline (1) the generation of a mutant library,
(2) the construction of a selection plasmid, (3) the selection procedure, and (4)
the identification and characterization of selected variants.
3.1. Generation of a Mutant Library
Random mutagenesis at the nucleotide level is a widespread strategy that targets
complete genes. The technique is usually applied if knowledge on the functional
contribution of defined protein motifs is limited, and if a complex structure-function
relationship is assumed. Random mutation is achieved either by
passing cloned genes through mutator strains (22,23) or by error-prone PCR
(11,12) (see Note 1). Our approach includes (a) the construction of an expression
vector containing the gene coding for T7 RNA polymerase and (b) random
mutagenesis by mutator strain passage.