Open Access Journals

AIMS Bioengineering, 2015, 2(3): 126-143. doi: 10.3934/bioeng.2015.3.126. Research article

Export file:

Format

RIS(for EndNote,Reference Manager,ProCite)
BibTex
Text

Content

Citation Only
Citation and Abstract

OneClick: A Program for Designing Focused Mutagenesis Experiments

Mark Warburton, Hossam Omar Ali, Wai Choon Liong, Arona Martin Othusitse, Amir Zaki Abdullah Zubir, Steve Maddock, Tuck Seng Wong

1 Department of Computer Science, The University of Sheffield, Regent Court, 211 Portobello, Sheffield S1 4DP, England;
2 ChELSI Institute and Advanced Biomanufacturing Centre, Department of Chemical and Biological Engineering, The University of Sheffield, Mappin Street, Sheffield S1 3JD, England;
3 A*STAR Institute of Materials Research and Engineering (IMRE), 3 Research Link, Singapore 117602;
4 School of Clinical Dentistry, The University of Sheffield, 19 Claremont Crescent, Sheffield S10 2TA, England.

Received date: , Accepted date: , Published date:

Abstract Full Text(HTML) Figure/Table Related pages

OneClick is a user-friendly web-based program, developed specifically for quick-and-easy design of focused mutagenesis experiments (e.g., site-directed mutagenesis and saturation mutagenesis). Written in Perl and developed into a web application using CGI programming, OneClick offers a step-by-step experimental design, from mutagenic primer design to analysis of a mutant library. Upon input of a DNA sequence encoding the protein of interest, OneClick designs the mutagenic primers according to user input, e.g., amino acid position to mutate, type of amino acid substitutions (e.g., substitution to a group of amino acids with similar chemical property) and type of mutagenic primers. OneClick has incorporated an extensive range of commercially available plasmids and DNA polymerases suitable for focused mutagenesis. Therefore, OneClick also provides information on PCR mixture preparation, thermal cycling condition, expected size of PCR product and agar plate to use during bacterial transformation. Importantly, OneClick also carries out a statistical analysis of the resultant mutant library, information of which is important for selection/screening. OneClick is a unique and invaluable tool in the field of protein engineering, allowing for systematic construction of a mutant library or a protein variant and simplifying molecular biology work. The program will be constantly updated to reflect the rapid development in the fields of molecular biology and protein engineering.

Figure/Table

Supplementary

Article Metrics

Keywords: protein engineering; enzyme engineering; directed evolution; biocatalysis; focused mutagenesis; site-directed mutagenesis (SDM); saturation mutagenesis; QuikChange

Citation: Mark Warburton, Hossam Omar Ali, Wai Choon Liong, Arona Martin Othusitse, Amir Zaki Abdullah Zubir, Steve Maddock, Tuck Seng Wong. OneClick: A Program for Designing Focused Mutagenesis Experiments. AIMS Bioengineering, 2015, 2(3): 126-143. doi: 10.3934/bioeng.2015.3.126

References:

1. Dalbadie-McFarland G, Cohen LW, Riggs AD, et al. (1982) Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function. Proc Natl Acad Sci U S A 79: 6409-6413.
2. Sigal IS, Harwood BG, Arentzen R (1982) Thiol-beta-lactamase: replacement of the active-site serine of RTEM beta-lactamase by a cysteine residue. Proc Natl Acad Sci U S A 79: 7157-7160.
3. Winter G, Fersht AR, Wilkinson AJ, et al. (1982) Redesigning enzyme structure by site-directed mutagenesis: tyrosyl tRNA synthetase and ATP binding. Nature 299: 756-758.
4. Brannigan JA, Wilkinson AJ (2002) Protein engineering 20 years on. Nat Rev Mol Cell Biol 3: 964-970.
5. Tee KL, Wong TS (2013) Polishing the craft of genetic diversity creation in directed evolution. Biotechnol Adv 31: 1707-1721.
6. Wong TS, Zhurina D, Schwaneberg U (2006) The diversity challenge in directed protein evolution. Comb Chem High Throughput Screen 9: 271-288.
7. Wong TS, Roccatano D, Zacharias M, et al. (2006) A statistical analysis of random mutagenesis methods used for directed protein evolution. J Mol Biol 355: 858-871.
8. Verma R, Wong TS, Schwaneberg U, et al. (2014) The Mutagenesis Assistant Program. Methods Mol Biol 1179: 279-290.
9. Reetz MT, Kahakeaw D, Lohmer R (2008) Addressing the numbers problem in directed evolution. Chembiochem 9: 1797-1804.
10. Edelheit O, Hanukoglu A, Hanukoglu I (2009) Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies. BMC Biotechnol 9: 61.
11. Wang W, Malcolm BA (1999) 2-stage PCR protocol allowing introduction of multiple mutations, deletions and insertions using QuikChange Site-Directed Mutagenesis. Biotechniques 26: 680-682.
12. Kibbe WA (2007) OligoCalc: an online oligonucleotide properties calculator. Nucleic Acids Res 35: W43-46.
13. Marmur J, Doty P (1962) Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 5: 109-118.
14. Wallace RB, Shaffer J, Murphy RF, et al. (1979) Hybridization of synthetic oligodeoxyribonucleotides to phi chi 174 DNA: the effect of single base pair mismatch. Nucleic Acids Res 6: 3543-3557.
15. Bernhardt R, Urlacher VB (2014) Cytochromes P450 as promising catalysts for biotechnological application: chances and limitations. Appl Microbiol Biotechnol 98: 6185-6203.
16. Munro AW, Leys DG, McLean KJ, et al. (2002) P450 BM3: the very model of a modern flavocytochrome. Trends Biochem Sci 27: 250-257.
17. Chen CK, Shokhireva T, Berry RE, et al. (2008) The effect of mutation of F87 on the properties of CYP102A1-CYP4C7 chimeras: altered regiospecificity and substrate selectivity. J Biol Inorg Chem 13: 813-824.
18. Vottero E, Rea V, Lastdrager J, et al. (2011) Role of residue 87 in substrate selectivity and regioselectivity of drug-metabolizing cytochrome P450 CYP102A1 M11. J Biol Inorg Chem 16: 899-912.
19. Graham-Lorence S, Truan G, Peterson JA, et al. (1997) An active site substitution, F87V, converts cytochrome P450 BM-3 into a regio- and stereoselective (14S,15R)-arachidonic acid epoxygenase. J Biol Chem 272: 1127-1135.
20. Li QS, Ogawa J, Schmid RD, et al. (2001) Residue size at position 87 of cytochrome P450 BM-3 determines its stereoselectivity in propylbenzene and 3-chlorostyrene oxidation. FEBS Lett 508: 249-252.
21. Oliver CF, Modi S, Sutcliffe MJ, et al. (1997) A single mutation in cytochrome P450 BM3 changes substrate orientation in a catalytic intermediate and the regiospecificity of hydroxylation. Biochemistry 36: 1567-1572.
22. Li QS, Ogawa J, Shimizu S (2001) Critical role of the residue size at position 87 in H2O2- dependent substrate hydroxylation activity and H2O2 inactivation of cytochrome P450BM-3. Biochem Biophys Res Commun 280: 1258-1261.
23. Kuper J, Tee KL, Wilmanns M, et al. (2012) The role of active-site Phe87 in modulating the organic co-solvent tolerance of cytochrome P450 BM3 monooxygenase. Acta Crystallogr Sect F Struct Biol Cryst Commun 68: 1013-1017.
24. Kuper J, Wong TS, Roccatano D, et al. (2007) Understanding a mechanism of organic cosolvent inactivation in heme monooxygenase P450 BM-3. J Am Chem Soc 129: 5786-5787.
25. Wong TS, Arnold FH, Schwaneberg U (2004) Laboratory evolution of cytochrome p450 BM-3 monooxygenase for organic cosolvents. Biotechnol Bioeng 85: 351-358.
26. Volkman BF, Liu TY, Peterson FC (2009) Chapter 3. Lymphotactin structural dynamics. Methods Enzymol 461: 51-70.
27. Reetz MT, Wang LW, Bocola M (2006) Directed evolution of enantioselective enzymes: iterative cycles of CASTing for probing protein-sequence space. Angew Chem Int Ed Engl 45: 1236-1241.

show all references

This article has been cited by:

1. Kang Lan Tee, Tuck Seng Wong, , Directed Enzyme Evolution: Advances and Applications, 2017, Chapter 8, 201, 10.1007/978-3-319-50413-1_8
2. Abdulrahman H. A. Alessa, Kang Lan Tee, David Gonzalez-Perez, Hossam E. M. Omar Ali, Caroline A. Evans, Alex Trevaskis, Jian-He Xu, Tuck Seng Wong, Accelerated directed evolution of dye-decolorizing peroxidase using a bacterial extracellular protein secretion system (BENNY), Bioresources and Bioprocessing, 2019, 6, 1, 10.1186/s40643-019-0255-7

Reader Comments

your name: * your email: *

Copyright Info: 2015, Tuck Seng Wong, et al., licensee AIMS Press. This is an open access article distributed under the terms of the Creative Commons Attribution Licese (http://creativecommons.org/licenses/by/4.0)

Download full text in PDF

Associated material

Metrics