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Site-saturation Mutagenesis: A Powerful Tool for Structure-Based Design of Combinatorial Mutation Libraries

UNIT 26.6

Evangelia G. Chronopoulou1 and Nikolaos E. Labrou1 1

Agricultural University of Athens, Athens, Greece

ABSTRACT This unit describes a method for site-saturation mutagenesis (SSM) using PCR amplification with degenerate synthetic oligonucleotides as primers. SSM allows the substitution of predetermined protein sites against all twenty possible amino acids at once. Therefore, SSM is a powerful approach in protein engineering to characterize structure-function relationships, as well as to create improved protein variants. The procedure accepts double-stranded plasmid isolated from the dam+ E. coli strain. The procedure is simple, fast, efficient, and eliminates time-consuming subcloning and ligation steps. Curr. C 2011 by John Wiley & Sons, Inc. Protoc. Protein Sci. 63:26.6.1-26.6.10. Keywords: protein engineering r mutagenesis r combinatorial mutation libraries

INTRODUCTION Protein engineering using site-specific or random mutagenesis offers the ability to characterize the relationship between protein structure and function (Jäckel et al., 2008; Gerlt and Babbitt, 2009; Goltermann et al., 2010; Labrou, 2010). Altering or deleting a gene’s wild-type sequence through in vitro molecular evolution strategies (random mutagenesis and high-throughput screening) is the method of choice for studying protein function and for creating new or novel sequences with improved or novel properties (Romero and Arnold, 2009; Dougherty and Arnold, 2009; Goltermann et al., 2010; Labrou, 2010). Directed protein evolution is accomplished by generating random or targeted mutations in the protein coding sequence and screening the library of protein variants for functional alterations (Fig. 26.6.1). This unit describes a method for site-saturation mutagenesis (SSM) using PCR amplification with degenerate synthetic oligonucleotides as primers (Georgescu et al., 2003; Zheng et al., 2004; Kotzia et al., 2007; Andreadeli et al., 2008; Kotzia and Labrou, 2009). The procedure is simple, fast, and efficient. Once a specific amino acid position within a protein sequence has been identified to be critical for the protein’s function, it is of great interest to determine the ideal amino acid residue for this position. SSM allows the substitution of specific sites against all twenty possible amino acids at once (Patrick and Firth, 2005; Andreadeli et al., 2008; Kotzia and Labrou, 2009). The procedure accepts double-stranded plasmid isolated from the dam+ E. coli strain. After completing PCR, the nonmutagenized parental DNA is degraded using restriction endonuclease DpnI. The final step is a transformation. This simple three-step protocol eliminates time-consuming subcloning, ligation, and single-stranded DNA rescue (Wang et al., 2007; Tseng et al., 2008).

Protein Engineering Current Protocols in Protein Science 26.6.1-26.6.10, February 2011 Published online February 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/0471140864.ps2606s63 C 2011 John Wiley & Sons, Inc. Copyright

26.6.1 Supplement 63

parent gene site-saturation mutagenisis

* * *

site-saturation mutants

library of site-saturation mutants

Figure 26.6.1 The main experimental steps of an in vitro–directed evolution process using site-saturation mutagenesis. A screening or selection procedure is employed to isolate the transformants with the desired activity.

BASIC PROTOCOL

SITE-SATURATION MUTAGENESIS SSM may be efficiently carried out using a protocol based on PCR (Fig. 26.6.2). With this protocol, two oligonucleotide primers containing mutant codon(s) with a mismatched sequence, complementary to the opposite strands of a double-stranded DNA plasmid template, are extended using PCR and DNA polymerase. The entire plasmid (nicked vector containing the mutated gene) is synthesized by DNA polymerase. The wild-type DNA plasmid template is then selectively degraded using DpnI endonuclease digestion. DpnI recognizes the sequence Gm ATC and cuts between the A and T to generate bluntended fragments. DpnI requires methylation of adenine residues for activity and thus digests only Gm ATC sequences containing N6 -methyladenine. Unlike the parent plasmid, the newly synthesized mutant genes do not contain methylated DNA and they therefore are not cleaved. The synthetic plasmid is then transformed directly into competent E. coli cells and the nick in the DNA is repaired in vivo by the cell machinery to yield a mutated, circular plasmid.

Site-saturation Mutagenesis


Current Protocols in Protein Science

Figure 26.6.2 mutations.

Whole plasmid, site-saturation mutagenesis using PCR. The symbols “*” represent

Materials E. coli competent cells (e.g., Top-10, XL-1 Blue, DH5 alpha; Stratagene, Invitrogen, Promega) LB medium (see recipe) Mini-prep solution I (see recipe) Mini-prep solution II (see recipe) Ice Mini-prep solution III (see recipe) Isopropanol Ethanol Sterile ddH2 O or TE buffer (APPENDIX 2A) High-fidelity Pfu DNA polymerase for PCR (e.g., Stratagene, Promega) 10× Pfu DNA polymerase buffer 10 mM dNTPs DpnI restriction endonuclease (e.g., New England Biolabs) LB plates with appropriate antibiotics (see recipe) 15-ml tubes (with caps) 37◦ C shaking incubator 2-ml microcentrifuge tubes Microcentrifuge Paper towels Vortex PCR tubes Thermal cycler Additional reagents and equipment for designing custom-synthesized oligonucleotides (primers) for PCR (APPENDIX 4J), analyzing the PCR product on an agarose gel (Irwin and Janssen, 2001; Wilson, 2002; also see APPENDIX 4F), and preparing miniprep plasmid DNA by alkaline lysis (APPENDIX 4C)

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Prepare the template plasmid DNA 1. Prepare the template plasmid DNA from a dam+ Escherichia coli strain (e.g., Top-10, XL-1 Blue, DH5 alpha). The preparation of plasmid DNA for subsequent use in polymerase chain reaction (PCR) methods can be achieved using a variety of protocols. For example, Qiagen (or equivalent) kits, standard alkaline lysis (APPENDIX 4C), or CsCl methods (Wilson, 1997) are acceptable.

Harvest bacterial culture for plasmid isolation 2. Inoculate 2 to 3 ml sterile LB medium (containing the appropriate antibiotic selective for the bacteria) with 10 μl bacterial stock culture in a loosely capped 15-ml tube. Incubate overnight on a shaker at 37◦ C with vigorous shaking. 3. Transfer 1.5 ml of the overnight culture into a 2-ml microcentrifuge tube. Microcentrifuge for 60 sec at high speed (12,000 × g), 4◦ C. Remove the supernatant using a pipet or aspirator, leaving the bacterial pellet as dry as possible.

Lyse the bacterial culture 4. Resuspend the bacterial pellet in 200 μl of mini-prep solution I. Resuspend the pellet completely by pipetting up and down or by vortexing. 5. Add 200 μl of mini-prep solution II (freshly prepared). Close the tube tightly and mix by inverting the tube gently (3 to 4 times). Do not vortex. 6. Leave the tube on ice and add 200 μl of mini-prep solution III. Mix by inverting the tube gently (3 to 4 times) to disperse solution III through the bacterial lysate. A white precipitate will form. Leave the tube on ice for 3 to 5 min. 7. Microcentrifuge the tube 10 min at high speed (12,000 × g), 4◦ C. Transfer the supernatant to a fresh microcentrifuge tube. Do not transfer any of the white pellets. 8. Add 0.7 vol (∼600 μl) of 100% (v/v) isopropanol to the supernatant. Mix well by inverting the tube several times. Leave the tube on ice for 5 min. Microcentrifuge 20 min at high speed (12,000 × g), 4◦ C. Remove and discard the supernatant. Stand the open tube in an inverted position on a paper towel to allow all of the fluid to drain away. With a pipet, remove any drops of fluid adhering to the walls of the tube. 9. To the DNA pellet, add 100 μl of ice-cold 75% (v/v) ethanol to wash. Vortex gently and centrifuge again for 5 min at high speed (12,000 × g), 4◦ C. Remove and discard the supernatant. Be sure that all of the ethanol has been removed from the pellet. If needed, air dry the pellet 10 to 20 min at room temperature. 10. Resuspend the pellet in 50 μl of sterile ddH2 O or TE buffer. Store at −20◦ C for 1 year or longer. A 5-μl sample should be sufficient to see clear bands on an agarose gel (APPENDIX 4F).

Design forward and reverse primers 11. Design the custom-synthesized primers for PCR (APPENDIX 4J). Two oligonucleotide primers (forward and reverse primer) are needed. The primers should be complementary to the opposite strands of the plasmid template, containing the mutant sequence flanked by 10 to 12 bases on each side. Codon randomization may be employed: NNN-, NNB-, NNK- or NNS-containing oligonucleotides (N = A/C/G/T; B = C/G/T; K = G/T; S = G/C). For example, working with the following sequence in a gene in a plasmid: caa tcg aga tcg aga ttc gca gag cgc ccg tgc tgg gaa gca gca tgg ctt acc gcg aga cgg gtg cgca Site-saturation Mutagenesis

and the goal is to randomize the condon “tgg” to NNN:



caa tcg aga tcg aga ttc gca gag cgc ccg tgc NNN gaa gca gca tgg ctt acc gcg aga cgg gtg cgca One primer will be: Primer no. 1: 5 -gca gag cgc ccg tgc NNN gaa gca gca tgg ctt-3 and the other primer will be the exact complement: Primer no. 2: 5 -aag cca tgc tgc ttc NNN gca cgg gcg ctc tgc-3 .

Run PCR-based SSM 12. Prepare a 50-μl PCR reaction with a proof-reading, non-displacing polymerase, such as Pfu DNA polymerase. Into a PCR tube, add the following: 5 to 20 ng DNA template (plasmid) (∼0.5 pmol) 5.0 μl 10× Pfu DNA polymerase buffer (including MgCl2 ) 0.5 μl 25 μM primer no. 1 (step 11) 0.5 μl 25 μM primer no. 2 (step 11) 1.0 μl 10 mM dNTP 1.0 μl (2.5 U) Pfu DNA polymerase Add ddH2 O to 50 μl. 13. Using a thermal cycler, run PCR using the following conditions: 1 cycle:

30 sec

15 to 20 cycles:

30 sec 1 min 2 min/kb of plasmid length

95◦ C (initial denaturation) 95◦ C (denatuation) 55◦ to 65◦ C (annealing) 72◦ C (extension).

The temperature for the annealing depends on the Tm of the primers, usually 5◦ C below the Tm of the primer. There are several programs available to predict the Tm for a given primer. For example, the programs Primer3 (http://frodo.wi.mit.edu/primer3/) and Primer3Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) work very well.

14. Following temperature cycling, place the reaction on ice for 2 min to cool the reaction.

Analyze the PCR product by gel electrophoresis 15. Analyze the PCR product (10 to 15 μl) on an agarose gel (Irwin and Janssen, 2001; Wilson, 2002; APPENDIX 4F; see Fig. 26.6.3). There should be a band corresponding to the expected product. In general, if the expected PCR product is visible, site-saturation mutagenesis will almost certainly work. Even if the product is not visible, the rest of the protocol can still be performed, but there may not be any colonies.

Digest the template DNA with DpnI 16. Add 1 μl of DpnI restriction enzyme (10 U/μl) directly to the amplification reaction. 17. Gently and thoroughly mix each reaction mixture by pipetting the solution up and down several times. Spin down the reaction mixtures in a microcentrifuge for 10 sec. 18. Incubate the tube 1 hr at 37◦ C. 19. DpnI digest the control PCR reaction (repeat steps 16 to 18).

Transform the plasmid into E. coli Transform the circular nicked DNA into a highly competent strain, such as E. coli XL1Blue, as described below. These cells will repair the nicks and not restrict the unmodified

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Figure 26.6.3 Analysis of PCR products after site-saturation mutagenesis reaction of an enzyme gene (approximately 600 bp).

product DNA. Also, perform transformation for the control reaction. No colonies should be observed. 20. Place highly competent XL1-Blue (100 to 200 μl) cells (1 × 108 /μg efficiency or greater) on ice. 21. Add 5 to 20 μl of DpnI-treated PCR reaction. Mix by tapping and immediately place on ice. Do not vortex. 22. Incubate on ice 15 to 20 min. 23. Heat shock 60 to 90 sec (42◦ C), and return to ice for 3 min. Do not exceed 42◦ C. 24. Add 1 ml LB medium. Incubate 1 hr at 37◦ C. 25. Plate a 20- to 100-μl aliquot on a prewarmed LB plate with appropriate antibiotic. Allow the plate to dry and incubate inverted at 37◦ C overnight (14 to 16 hr). 26. Select six single colonies. Prepare miniprep plasmid DNA by alkaline lysis (APPENDIX 4C). 27. Submit the isolated plasmid for sequencing to confirm mutations.

REAGENTS AND SOLUTIONS Use Milli-Q-purified water or equivalent in all recipes and protocol steps. For common stock solutions, see APPENDIX 2E; for suppliers, see SUPPLIERS APPENDIX.

LB broth (per liter) 10 g tryptone 5 g yeast extract 5 g NaCl 1 ml 1 M NaOH (pH should be approximately 7.5) Autoclave Site-saturation Mutagenesis

When the medium is sufficiently cooled (about 60◦ C) add the desired antibiotic(s) and mix thoroughly.



LB plates (per liter) 10 g tryptone 5 g yeast extract 5 g NaCl 15 g bacteriological agar 1 ml 1 M NaOH (pH should be approximately 7.5) Autoclave When the medium is sufficiently cooled (about 60◦ C) add the desired antibiotic(s) and mix thoroughly. Add 15 to 20 ml of the molten agar mixture per 100-mm diameter petri dish and allow to set at room temperature before storage. Remove excess moisture by allowing the plates to dry near the Bunsen flame.

Mini-prep solution I (100 ml) 25 mM Tris·Cl, pH 8.0 (APPENDIX 2E) 50 mM glucose 10 mM EDTA RNase A (10 mg/100 ml) Autoclave (add filter-sterilized RNase after autoclaving) Store up to 1 month at 4◦ C Mini-prep solution II (100 ml) To 80 ml ddH2 O add: 10 ml 10% SDS (sodium dodecyl sulfate, no need to sterilize; 1% final) 10 ml 2 M NaOH (200 mM final concentration) Prepare fresh If the SDS precipitates (as it may on cold days), warm it gently in a water bath until it dissolves.

Mini-prep solution III (100 ml) 60 ml of 5 M potassium acetate 11.5 ml glacial acetic acid 28.5 ml ddH2 O Autoclave Store up to 1 month at 4◦ C The final solution is 3 M potassium and 5 M acetate; the pH should be 5.5.

COMMENTARY Background Information Protein engineering and directed evolution are powerful tools for the investigation of the relationship between structure and function of proteins and enzymes and have important biotechnological applications in the design of new or novel functions (e.g., enzyme activities; Jäckel et al., 2008; Gerlt and Babbitt, 2009; Goltermann et al., 2010; Labrou, 2010). There are two main approaches for protein engineering. The first is known as rational design, and uses detailed knowledge of the structure and function of the protein to design specific changes into the protein structure (Otten et al., 2010). The major drawback of this approach is that detailed structural knowledge of

a protein is often unavailable, and even when it is available, it can be extremely difficult to predict the effects of various mutations. The second strategy is known as directed evolution. In this approach, random mutagenesis is applied to a protein, and a selection regime is used to select protein variants that have desired properties (Dougherty and Arnold, 2009; Romero and Arnold, 2009; Goltermann et al., 2010; Labrou, 2010). Once a specific amino acid position within a protein sequence has been identified to be critical for the protein’s function, it is of great interest to determine the ideal amino acid residue for this position (Kotzia et al., 2007; Andreadeli et al., 2008; Kotzia and Labrou, 2009). SSM is suited

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for this application and is used to explore additional pathways and enable rapid diversification in protein traits. For example, Kotzia and Labrou (2009) used in vitro–directed evolution for the creation of L-asparaginase mutant enzyme with improved stability. A library of enzyme variants was created by directed evolution and after screening, a thermostable variant was identified that contained a singlepoint mutation (Asp133Val). Site-saturation mutagenesis at position 133 was used to further analyze the role of the amino acid at this position on the thermostability of the enzyme. Screening of a library of random Asp133 mutants confirmed that this position significantly influences the enzyme’s thermostability and showed that the mutant Asp133Leu exhibits the optimal thermostability. The simple three-step protocol described in this unit eliminates time-consuming subcloning, ligation, and single-stranded DNA rescue. This protocol has been utilized to generate several site-saturation libraries with a success rate of >95%. For example, Andreadeli et al. (2008) used this protocol to alter the coenzyme specificity of the enzyme formate dehydrogenase from C. boidinii (CboFDH). This enzyme catalyzes the oxidation of formate anion to carbon dioxide with concomitant reduction of NAD+ to NADH. CboFDH is highly specific to NAD+ and does not accept NADP+ . The authors carried out two rounds of site-saturation mutagenesis and screening and identified a double mutant Asp195Gln/Tyr196His, which exhibited more than 2×107 -fold improvement in overall catalytic efficiency with NADP+ and more than 900-fold decrease in the efficiency with NAD+ as cofactors. Saturation mutagenesis may also be achieved using a protocol that involves two mutagenic primers (primer 1 and 2) and two additional primers (primer 3 and 4) complementary at the 5 and 3 end of the gene. Each primer is used in a separate reaction (separate tubes, same conditions) to generate two halves of the region in two separate reactions and put them together in the next step where they anneal in the 25- to 30-bp region of complementarity and prime off each other to yield the full length product. Other methods of saturation mutagenesis that have been developed are: codon cassette mutagenesis (Kegler-Ebo et al., 1994), iterative saturation mutagenesis (Reetz and Carballeira, 2007; Reetz et al., 2009), including combinatorial cassette mutagenesis (Reidhaar-Olson and Sauer, 1988), recursive ensemble mutagenesis (Arkin and

Youvan, 1992), scanning saturation mutagenesis (Maynard et al., 2002), and sequence saturation mutagenesis (Wong et al., 2004).

Critical Parameters Incorporating the full degeneracy (using NNN codons, N = A/C/G/T) of the genetic code may give a library with a significant percentage of the genes containing premature termination codons (stop codons). In addition, the most common variants (with combinations of Arg, Leu, and Ser, each of which are encoded by six codons) will be very abundant compared to the rarest (with Met or Trp at each randomized position), which complicates the screening or selection procedures. By reducing codon sets, these problems may be alleviated. For example: NNB codons (B = C/G/T) have the smallest probability of encoding a stop codon (one in 48), while NNK and NNS codons (K = G/T; S = G/C)) minimize the overrepresentation of the commonest variants (Patrick and Firth, 2005). The restriction endonuclease DpnI recognizes the sequence shown in Figure 26.6.4.

CH3 5′ 3′

G A T C C T A G

3′ 5′

CH3 Figure 26.6.4 Recognition sequence of the restriction endonuclease DpnI.

DNA isolated from almost all common strains of E. coli is Dam-methylated at the sequence 5 -GATC- 3. DpnI digestion results in the degradation of the in vivo–methylated parental plasmid DNA template and hybrid DNA. There is no need to carry out a ligation before the transformation. The PCR product is a “nicked” circle with nicks in opposite strands. This is identical to a classical de-phosphorylated vector plus insert transformation.

Troubleshooting Table 26.6.1 lists some of the common problems that may be encountered using the protocols described in this unit. Explanations



Table 26.6.1 Troubleshooting Guide for Creating a Combinatorial Gene Library

Problem

Probable cause

Solution

No PCR product

PCR conditions and parameters not optimized (see APPENDIX 4J)

Optimize the cycling parameters. Mutagenesis of long DNA plasmids may require optimization of PCR conditions and amounts of template and primers. Redesign the primers. If one primer has a lower melting temperature (Tm ) due to a lower GC content, extend the primer length to increase the Tm . Analyze the plasmid DNA template on a gel to verify the quantity and quality. In the PCR reaction, most of the primers will be annealing to each other and only a small portion will be annealing to the target sequence on the plasmid. Hence, avoid normal Taq DNA polymerase. The primers must be FPLC-, HPSF-, or PAGE-gel purified. If you see a strong primer dimer band, it means that primer-primer annealing is favored over primer-template annealing. Try reducing the concentration of primer. Try repeating the protocol with 5% DMSO in the PCR reaction mixture. DMSO disrupts base pairing, facilitating strand separation in GC-rich regions of DNA and reducing the propensity of the DNA to form secondary structure.

Low mutagenesis efficiency and few colonies in the control reaction

DpnI digestion did not work properly

Increase the amount of the DpnI in the reaction (e.g., 4 μl) or extend the incubation time (2 hr). Verify that the template DNA was isolated from a dam+ E. coli strain.

Low transformation efficiency

The cells were handled improperly There were impurities in the DNA

Test the efficiency of the competent cells using a control plasmid (e.g., pUC18). Do not vortex the cells. Refreezing can decrease transformation efficiency. Remove impurities from the PCR product by ethanol precipitation (Moore and Dowhan, 2002)

Mutant error

Primer quality

Check the quality (e.g., purity) of the synthesized primers

of possible causes of the problems and suggested approaches for overcoming these problems are included.

Anticipated Results This protocol is highly effective and simple for making site-directed mutants in plasmids. Plasmids 95%.

Time Considerations In general, the protocol (DNA purification, PCR, agarose gel analysis, DpnI digestion, and transformation) may be completed within a single working day.

Literature Cited Andreadeli, A., Platis, D., Tishkov, V., Popov, V., and Labrou, N.E. 2008. Structure-guided alteration of coenzyme specificity of formate dehydrogenase by saturation mutagenesis to enable efficient utilization of NADP+ . FEBS J. 275:3859-3869. Arkin, A.P. and Youvan, D.C. 1992. A combinatorial optimization procedure for protein engineering: Simulation of recursive ensemble mutagenesis. Proc. Natl. Acad. Sci. U.S.A. 89:7811-7815. Dougherty, M.J. and Arnold, F.H. 2009. Directed evolution: New parts and optimized function. Curr. Opin. Biotechnol. 20:486-491. Georgescu, R., Bandara, G., and Sun, L. 2003. Saturation mutagenesis. In Methods in Molecular Biology Vol. 231: Directed Evolution Library Creation (F.H. Arnold and G. Georgiou, eds.) pp. 75-83. Humana Press, Totowa, N.J. Gerlt, A. and Babbitt, P.C. 2009 Enzyme (re)design: Lessons from natural evolution and computation. Curr. Opin. Chem. Biol. 13:10-18. Protein Engineering


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Goltermann, L., Larsen, M.S., Banerjee, R., Joerger, A.C., Ibba, M., and Bentin, T. 2010. Protein evolution via amino acid and codon elimination. PLoS One 26:e10104. Irwin, N. and Janssen, K.A. 2001. Gel Electrophoresis of DNA and Pulsed-field Agarose Gel Electrophoresis. Molecular Cloning Vol. 1: A Laboratory Manual (J. Sambrook and D.W. Russell, eds.) pp. 5.1-5.17. CSHL Press, New York. Jäckel, C., Kast, P., and Hilvert, D. 2008. Protein design by directed evolution. Annu. Rev. Biophys. 37:153-173. Kegler-Ebo, D.M., Docktor, C.M., and DiMaio, D. 1994. Codon cassette mutagenesis: A general method to insert or replace individual codons by using universal mutagenic cassettes. Nucleic Acids Res. 22:1593-1599. Kotzia, G.A. and Labrou, N.E. 2009. Engineering thermal stability of L-asparaginase by in vitro directed evolution. FEBS J. 276:1750-1761. Kotzia, G.A.,Lappa, K., and Labrou, N.E. 2007. Tailoring structure-function properties of Lasparaginase: Engineering resistance to trypsin cleavage. Biochem. J. 404:337-343. Labrou, N.E. 2010. Random mutagenesis methods for in vitro directed enzyme evolution. Curr. Protein Pept. Sci. 11:91-100. Maynard, J.A., Chen, G., Georgiou, G., and Iverson, B.L. 2002. In vitro scanning–saturation mutagenesis. Methods Mol. Biol. 182:149-163. Moore, D. and Dowhan, D. 2002. Purification and concentration of DNA from aqueous solutions. Curr. Protoc. Mol. Biol. 59:2.1.1-2.1.10. Otten, L.G., Hollmann, F., and Arends, I.W. 2010. Enzyme engineering for enantioselectivity: From trial-and-error to rational design? Trends Biotechnol. 28:46-54. Patrick, W.M. and Firth, A.E. 2005. Strategies and computational tools for improving randomized protein libraries. Biomol. Eng. 22:105-112. Reetz, M.T. and Carballeira, J.D. 2007. Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nat. Protoc. 2:891-903. Reetz, M.T., Kahakeaw, D., and Sanchis, J. 2009. Shedding light on the efficacy of laboratory evo-

lution based on iterative saturation mutagenesis. Mol. Biosyst. 5:115-122. Reidhaar-Olson, J.F. and Sauer, R.T. 1988. Combinatorial cassette mutagenesis as a probe of the informational content of protein sequences. Science 241:53-58. Romero, P.A. and Arnold, F.H. 2009. Exploring protein fitness landscapes by directed evolution. Nat. Rev. Mol. Cell Biol. 10:866-876. Tseng, W.C., Lin. J.W., Wei, T.Y., and Fang, T.Y. 2008. A novel megaprimed and ligase-free, PCR-based, site-directed mutagenesis method. Anal. Biochem. 375:376-378. Wang, J., Zhang, S., Tan, H., and Zhao, Z.K. 2007. PCR-based strategy for construction of multisite-saturation mutagenic expression library. J. Microbiol. Methods 71:225-230. Wilson, K. 1997. Preparation of genomic DNA from bacteria. Curr. Protoc.Mol. Biol. 27:2.4.1-2.4.5. Wilson, K. 2002. Preparation and analysis of DNA: Agarose gel electrophoresis. In Current Protocols in Molecular Biology Vol. 1: Short Protocols In Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 2.13-2.14. John Wiley & Sons, Hoboken. Wong, T.S., Tee, K.L., Hauer, B., and Schwaneberg, U. 2004. Sequence saturation mutagenesis (SeSaM): A novel method for directed evolution. Nucleic Acids Res. 32:1-8. Zheng, L., Baumann, U., and Reymond, J.L. 2004. An efficient one-step sitedirected and sitesaturation mutagenesis protocol. Nucleic Acids Res. 32:1-5.

Internet Resources www.insilico.uni-duesseldorf.de When using the tool in your experiments, use the following citation: Ulrich Krauss and Thorsten Eggert (2005) Insilico.mutagenesis: a primer selection tool designed for sequence scanning applications used in directed evolution experiments. BioTechniques 39 (5): p679-682. http://www.bioinformatics.org/primerx/ PrimerX: Automated design of mutagenic primers for site-directed mutagenesis.


