Abstract
Methods for the cloning of large numbers of open reading frames (ORFs) into expression vectors are of critical importance for diverse disciplines in biology. Here I describe a system termed FX cloning that facilitates the high-throughput generation of expression constructs. FX cloning combines attractive features of established recombination- and single-strand-annealing-based cloning methods that were thus far not unified in one single method. FX cloning allows the straightforward transfer of a sequence-verified ORF to a variety of expression vectors, and it avoids the common but undesirable feature of significantly extending target ORFs with cloning-related sequences. It leaves a minimal seam of only a single amino acid to either side of the protein. Furthermore, FX cloning is highly efficient and economic in its use. The method is based on a class IIS restriction enzyme and negative selection markers. The full procedure takes place in one pot and does not require intermediate purifications. The method has proven to be very robust and suitable for all common pro- and eukaryotic expression systems.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Blommel PG, Martin PA, Wrobel RL, Steffen E, Fox BG (2006) High efficiency single step production of expression plasmids from cDNA clones using the Flexi Vector cloning system. Protein Expr Purif 47:562–570
Hartley JL, Temple GF, Brasch MA (2000) DNA cloning using in vitro site-specific recombination. Genome Res 10:1788–1795
Aslanidis C, de Jong PJ (1991) Coincidence cloning of Alu PCR products. Proc Natl Acad Sci U S A 88:6765–6769
Li MZ, Elledge SJ (2007) Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat Methods 4:251–256
Klock HE, Koesema EJ, Knuth MW, Lesley SA (2008) Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts. Proteins 71:982–994
Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345
Berrow NS, Alderton D, Owens RJ (2009) The precise engineering of expression vectors using high-throughput In-Fusion PCR cloning. Methods Mol Biol 498:75–90
Geertsma ER, Dutzler R (2011) A versatile and efficient high-throughput cloning tool for structural biology. Biochemistry 50:3272–3278
Szybalski W, Kim SC, Hasan N, Podhajska AJ (1991) Class-IIS restriction enzymes—a review. Gene 100:13–26
Bernard P, Gabant P, Bahassi EM, Couturier M (1994) Positive-selection vectors using the F plasmid ccdB killer gene. Gene 148:71–74
Recorbet G, Robert C, Givaudan A, Kudla B, Normand P, Faurie G (1993) Conditional suicide system of Escherichia coli released into soil that uses the Bacillus subtilis sacB gene. Appl Environ Microbiol 59:1361–1366
Zheng L, Baumann U, Reymond JL (2004) An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res 32:e115
Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS (1991) ‘Touchdown’ PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res 19:4008
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor, New York
Guzman LM, Belin D, Carson MJ, Beckwith J (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177:4121–4130
Tillett D, Neilan BA (1999) Enzyme-free cloning: a rapid method to clone PCR products independent of vector restriction enzyme sites. Nucleic Acids Res 27:e26
Marsischky G, LaBaer J (2004) Many paths to many clones: a comparative look at high-throughput cloning methods. Genome Res 14:2020–2028
Hamilton MD, Nuara AA, Gammon DB, Buller RM, Evans DH (2007) Duplex strand joining reactions catalyzed by vaccinia virus DNA polymerase. Nucleic Acids Res 35:143–151
Cohen SN, Chang AC, Boyer HW, Helling RB (1973) Construction of biologically functional bacterial plasmids in vitro. Proc Natl Acad Sci U S A 70:3240–3244
Colwill K, Wells CD, Elder K, Goudreault M, Hersi K, Kulkarni S, Hardy WR, Pawson T, Morin GB (2006) Modification of the creator recombination system for proteomics applications—improved expression by addition of splice sites. BMC Biotechnol 6:13
Liu Q, Li MZ, Leibham D, Cortez D, Elledge SJ (1998) The univector plasmid-fusion system, a method for rapid construction of recombinant DNA without restriction enzymes. Curr Biol 8:1300–1309
Li MZ, Elledge SJ (2005) MAGIC, an in vivo genetic method for the rapid construction of recombinant DNA molecules. Nat Genet 37:311–319
Acknowledgements
E.R.G. acknowledges a long-term fellowship from the Human Frontier Science Program and thanks Prof. Raimund Dutzler for critical reading of the manuscript.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer New York
About this protocol
Cite this protocol
Geertsma, E.R. (2013). FX Cloning: A Versatile High-Throughput Cloning System for Characterization of Enzyme Variants. In: Samuelson, J. (eds) Enzyme Engineering. Methods in Molecular Biology, vol 978. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-293-3_10
Download citation
DOI: https://doi.org/10.1007/978-1-62703-293-3_10
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-292-6
Online ISBN: 978-1-62703-293-3
eBook Packages: Springer Protocols