Abstract
The immense amount of gene sequences available nowadays allows scientist to screen broadly for extraordinary proteins. Reliable cloning tools that allow the parallel processing of many targets are vital for the success of this strategy. The FX cloning procedure detailed here is such a straightforward and efficient tool. It is dedicated to the cloning of open reading frames (ORFs) with the final aim of expressing the corresponding proteins. FX cloning combines attractive features of established high-throughput cloning methods that were thus far not unified in one single method. It facilitates the subcloning of a sequence-verified ORF to a variety of expression vectors, but is sufficiently versatile to accept PCR products as well. Moreover, the common, but undesirable feature of extending target ORFs with long cloning-related sequences is avoided. It leads to the addition of only one amino acid to each side of the protein. As a consequence, only one primer pair or PCR product suffices to generate expression vectors for both N- and C-terminal translational fusions. FX cloning is highly efficient and economical in its use. The method is suited for high-throughput cloning projects and also for everyday cloning of single targets. FX cloning is based on the use of type IIS restriction enzymes and negative selection markers. The full procedure takes place in one pot in less than 3 h and does not require intermediate purification steps nor extensive handling. The method has proven to be very robust and suitable for all common expression systems.
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References
Pagani I, Liolios K, Jansson J et al (2012) The Genomes OnLine Database (GOLD) v. 4: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res 40:D571–D579
Yang X, Boehm JS, Yang X et al (2011) A public genome-scale lentiviral expression library of human ORFs. Nat Methods 8:659–661
Mancia F, Love J (2011) High throughput platforms for structural genomics of integral membrane proteins. Curr Opin Struct Biol 21:517–522
Xiao R, Anderson S, Aramini J et al (2010) The high-throughput protein sample production platform of the Northeast Structural Genomics Consortium. J Struct Biol 172:21–33
Gräslund S, Nordlund P, Weigelt J et al (2008) Protein production and purification. Nat Methods 5:135–146
Hartley JL, Temple GF, Brasch MA (2000) DNA cloning using in vitro site-specific recombination. Genome Res 10:1788–1795
Zhang Y, Werling U, Edelmann W (2012) SLiCE: a novel bacterial cell extract-based DNA cloning method. Nucleic Acids Res 40:e55
Aslanidis C, de Jong PJ (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res 18:6069–6074
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
Thieme F, Engler C, Kandzia R et al (2011) Quick and clean cloning: a ligation-independent cloning strategy for selective cloning of specific PCR products from non-specific mixes. PLoS One 6:e20556
Li MZ, Elledge SJ (2007) Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat Methods 4:251–256
Gibson DG, Young L, Chuang R et al (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345
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
Geu-Flores F, Nour-Eldin HH, Nielsen MT et al (2007) USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products. Nucleic Acids Res 35:e55
Klock HE, Koesema EJ, Knuth MW et al (2008) Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts. Proteins 71:982–994
van den Ent F, Löwe J (2006) RF cloning: a restriction-free method for inserting target genes into plasmids. J Biochem Biophys Methods 67:67–74
Quan J, Tian J (2009) Circular polymerase extension cloning of complex gene libraries and pathways. PLoS One 4:e6441
Erijman A, Dantes A, Bernheim R et al (2011) Transfer-PCR (TPCR): a highway for DNA cloning and protein engineering. J Struct Biol 175:171–177
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 et al (1991) Class-IIS restriction enzymes–a review. Gene 100:13–26
Bernard P, Gabant P, Bahassi EM et al (1994) Positive-selection vectors using the F plasmid ccdB killer gene. Gene 148:71–74
Recorbet G, Robert C, Givaudan A et al (1993) Conditional suicide system of Escherichia coli released into soil that uses the Bacillus subtilis sacB gene. Appl Environ Microbiol 59:1361–1366
Casadaban MJ, Cohen SN (1980) Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol 138:179–207
Don RH, Cox PT, Wainwright BJ et al (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 Laboratory Press, Cold Spring Harbor, NY
Guzman LM, Belin D, Carson MJ et al (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177:4121–4130
Acknowledgments
E.R.G. acknowledges a long-term fellowship from the Human Frontier Science Program. Iwan Zimmermann and Margrit Mathys are thanked for critical comments on the manuscript. Mark Schmitz and Carlo Bertozzi are thanked for contributing to the FX cloning website.
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Geertsma, E.R. (2014). FX Cloning: A Simple and Robust High-Throughput Cloning Method for Protein Expression. In: Valla, S., Lale, R. (eds) DNA Cloning and Assembly Methods. Methods in Molecular Biology, vol 1116. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-764-8_11
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DOI: https://doi.org/10.1007/978-1-62703-764-8_11
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