Site‐Specific Recombinational Cloning Using Gateway and In‐Fusion Cloning Schemes

Jaehong Park1, Andrea L. Throop2, Joshua LaBaer2

1 Harvard Medical School, Cambridge, Massachusetts, 2 Biodesign Institute, Arizona State University, Tempe, Arizona
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 3.20
DOI:  10.1002/0471142727.mb0320s110
Online Posting Date:  April, 2015
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Abstract

The comprehensive study of protein structure and function, or proteomics, depends on the obtainability of full‐length cDNAs in species‐specific expression vectors and subsequent functional analysis of the expressed protein. Recombinational cloning is a universal cloning technique based on site‐specific recombination that is independent of the insert DNA sequence of interest, which differentiates it from classical restriction enzyme−based cloning methods. Recombinational cloning enables rapid and efficient parallel transfer of DNA inserts into multiple expression systems. This unit summarizes strategies for generating expression‐ready clones using the most popular commercial recombinational cloning technologies, Gateway (Life Technologies) and In‐Fusion (Clontech). © 2015 by John Wiley & Sons, Inc.

Keywords: recombinational cloning; Gateway; In‐Fusion; high‐throughput; Cre‐Lox

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: PCR Amplification of Target Genes
  • Basic Protocol 2: Capture of ORFS to Make Entry Clones for Gateway Cloning: The BP Reaction
  • Basic Protocol 3: Capture of ORFS to Make Entry Clones for In‐Fusion Cloning
  • Basic Protocol 4: Generation of Gateway Expression Clones
  • Alternate Protocol 1: Generation of Expression Clones Using the Cre‐Lox System
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: PCR Amplification of Target Genes

  Materials
  • 2.0 U/μl Phusion High‐Fidelity DNA polymerase and 5× reaction buffer (New England Biolabs)
  • 10 mM 4dNTPs
  • 100% dimethyl sulfoxide (DMSO)
  • PCR‐quality H 2O
  • 10 μM gene‐specific oligonucleotide primer solutions (individual 3′ and 5′ solutions) in sterile H 2O (for primer design, see protocol introduction)
  • DNA template: 10 ng/μl first‐strand cDNA or 1‐50 ng/μl plasmid DNA
  • 100 μM universal oligonucleotide primer solutions (individual 3′ and 5′ solutions)
  • PCR Clean‐Up kit (e.g., Qiagen or Macherey Nagel for high‐throughput)
  • 2× CloneAmp HiFi PCR Premix (Clontech; for In‐Fusion)
  • PCR tubes (thin‐walled) or 96‐well plates
  • Multichannel pipettor (optional)
  • Thermal cycler
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5) and for preparation (unit 1.6, optional) and quantitation ( appendix 3D) of DNA

Basic Protocol 2: Capture of ORFS to Make Entry Clones for Gateway Cloning: The BP Reaction

  Materials
  • 150 ng/μl entry vector (e.g., pDONR221; Life Technologies)
  • Gateway BP Clonase II enzyme mix (Life Technologies)
  • PCR‐quality H 2O
  • 10 to 50 ng/μl attB‐PCR product prepared using the Gateway scheme (see protocol 1)
  • Competent T1 phage−resistant (T1R) E. coli (e.g., One Shot MAX Efficiency DH5α competent cells; Life Technologies)
  • LB plates and liquid medium (unit 1.1) containing 50 μg/ml kanamycin (or other appropriate antibiotic)
  • 50% (v/v) glycerol, sterile
  • Plasmid DNA isolation kit (e.g., Qiagen or Macherey Nagel; optional)
  • 1.5‐ml microcentrifuge tubes or 96‐well plate
  • Multichannel pipettor (optional)
  • 25° and 37°C incubators
  • Sequence verification tools, e.g., BLAST (http://www.ncbi.nlm.nih.gov/blast) or Sequencher (http://www.genecodes.com/sequencher)
  • Additional reagents and equipment for bacterial transformation (unit 1.8), plasmid miniprep (unit 1.6; optional), and DNA sequencing

Basic Protocol 3: Capture of ORFS to Make Entry Clones for In‐Fusion Cloning

  Additional Materials (also see protocol 3)
  • 50 ng/μl entry vector DNA (e.g., pGWNcoEco; DNASU, https://dnasu.org/DNASU/)
  • Appropriate restriction enzymes and buffers (e.g., NcoI and Eco0109i for pGWNcoEco; New England Biolabs)
  • GelStar nucleic acid stain (Lonza)
  • Gel extraction kit (e.g., QIAquick Gel Extraction kit; Qiagen)
  • 5× In‐Fusion HD Enzyme premix and 10× reaction buffer (Clontech)
  • 500 ng/μl PCR product prepared for In‐Fusion cloning (see protocol 1)
  • Low‐frequency UV light box
  • Razor blades
  • PCR tubes or 96‐well plate (thin‐walled)
  • Thermal cycler
  • Additional reagents and equipment for restriction enzyme digestion (unit 3.1), agarose gel electrophoresis (unit 2.5), and determination of DNA concentration ( appendix 3D)

Basic Protocol 4: Generation of Gateway Expression Clones

  Materials
  • Expression vector with a different selectable marker than the entry clone
  • Appropriate restriction enzymes and buffers (e.g., New England Biolabs)
  • T4 DNA polymerase or Klenow fragment and buffer (e.g., New England Biolabs)
  • TE buffer, pH 8.0 ( appendix 22)
  • 1 U/μl T4 DNA ligase and 10× buffer (e.g., New England Biolabs)
  • 5 ng/μl Gateway recombination cassette (rfA, rfB, or rfC; Life Technologies)
  • Sterile H 2O
  • E. coli competent cell cultures (e.g., One Shot ccdB Survival 2 T1R and DH5α; Life Technologies)
  • LB plates and medium (unit 1.1) containing 30 μg/ml chloramphenicol (or other antibiotic appropriate for recombination cassette)
  • 50% (v/v) glycerol, sterile
  • Plasmid DNA isolation kit (e.g., Qiagen or Macherey Nagel; optional)
  • 150 ng/μl commercial Gateway destination (expression) vector (e.g., pDEST27; Life Technologies; optional)
  • Gateway LR Clonase II Enzyme mix (Life Technologies)
  • PCR‐quality H 2O
  • 150 ng/μl entry clone plasmid DNA (see protocol 3, protocol 4, or other plasmid resources, e.g., DNASU)
  • LB plates and medium containing 100 μg/ml ampicillin (or other antibiotic appropriate for expression vector)
  • 1.5‐ml microcentrifuge tubes
  • 96‐well plate (optional)
  • Multichannel pipettor (optional)
  • 25° and 37°C incubators
  • Additional reagents and equipment for restriction enzyme digestion (unit 3.1), generating blunt DNA ends (units 3.5 & 3.16), bacterial transformation (unit 1.8), plasmid DNA miniprep (unit 1.6; optional), sequencing (optional), and determination of DNA concentration (APPENDEX 3.NaN)

Alternate Protocol 1: Generation of Expression Clones Using the Cre‐Lox System

  Additional Materials (also see Basic Protocols protocol 11 and protocol 54)
  • LoxP primers (see step 1)
  • LoxP template (e.g., Addgene)
  • Appropriate restriction enzymes and buffers (New England Biolabs)
  • LB plates and medium (unit 1.1) containing 100 μg/ml kanamycin (or other appropriate antibiotic)
  • Cre‐Lox expression vector (e.g., pLP‐CMVneo, DNASU) or other expression vector to make Cre‐Lox‐compatible
  • Cre recombinase (New England Biolabs or purified in‐house according to Cantor and Chong, )
  • 10× Cre reaction buffer (New England Biolabs)
  • LB plates containing 30 μg/ml chloramphenicol (or other appropriate antibiotic) and 7% (w/v) sucrose
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Figures

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Literature Cited

Literature Cited
  Bernard, P. and Couturier, M. 1992. Cell killing by the F plasmid CcdB protein involves poisoning of DNA‐topoisomerase II complexes. J. Mol. Biol. 226:735‐745.
  Bird, L.E. 2011. High throughput construction and small scale expression screening of multi‐tag vectors in Escherichia coli. Methods 55:29‐37.
  Brizuela, L., Richardson, A., Marsischky, G., and LaBaer, J. 2002. The FLEXGene repository: Exploiting the fruits of the genome projects by creating a needed resource to face the challenges of the post‐genomic era. Arch. Med. Res. 33:318‐324.
  Cantor, E.J. and Chong, S. 2001. Intein‐mediated rapid purification of cre recombinase. Protein Expr. Purif. 22:135‐140.
  Ceroni, A., Sibani, S., Baiker, A., Pothineni, V.R., Bailer, S.M., LaBaer, J., Hass, J., and Campbell, C.J. 2010. Systematic analysis of the IgG antibody immune response against varicella zoster virus (VSV) using a self‐assembled protein microarray. Mol. BioSyst. 6:1604‐1610.
  Festa, F., Steel, J., Bian, X., and LaBaer, J., 2013. High‐throughput cloning and expression library creation for functional proteomics. Proteomics 13:1381‐1399.
  Gibson, D.G. 2011. Gene and genome construction in yeast. Curr. Protoc. Mol. Biol. 94:3.22.01‐3.22.17.
  Gibson, D.G., Young, L., Chuang, R.‐R., Venter, J.C., Hutchison, C.A. III, and Smith, H.O. 2009. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods. 6:343‐345.
  Hamilton, M.D., Nuara, A.A., Gammon, D.B., Buller, R.M., and Evans, D.H. 2006. Duplex strand joining reactions catalyzed by vaccinia virus DNA polymerase. Nucleic Acids Res. 35:143‐151.
  Hartley, J.L., Temple, G.F., and Brasch, M.A. 2000. DNA cloning using in vitro site‐specific recombination. Genome Res. 10:1788‐1795.
  Irwin, C.R., Farmer, A., Willer, D.O., and Evans, D.H. 2012. In‐Fusion® cloning with vaccinia virus DNA polyermase. In Vaccinia Virus and Poxvirology: Methods and Protocols, Vol. 890: Methods in Molecular Biology (S.N. Isaacs, ed.), pp. 23‐35. Humana Press, New York.
  Landy, A. 1989. Dynamic, structural, and regulatory aspects of lambda site‐specific recombination. Annu. Rev. Biochem. 58:913‐949.
  Liang, X., Peng, L., Baek C., and Katzen, R. 2013. Single step BP/LR combined Gateway reaction. Biotechniques 5:265‐268.
  Marsischky, G. and LaBaer, J. 2004. Many paths to many clones: A comparative look at high‐throughput cloning methods. Genome Res. 14:2020‐2028.
  Murthy, T., Rolfs, A., Hu, Y., Zhenwei, S., Raphael, J., Moreira, D., Kelley, F., McCarron, S., Jepson, D., Taycher, E., Zuo, D., Mohr, S.E., Fernadez, M., Brizuela, L., and LaBaer, J. 2007. A full‐genomic sequence‐verified protein‐coding collection for Francisella tularensis. PLoS One. 2:e577.
  Nash, H.A. 1977. Integration and excision of bacteriophage lambda. Curr. Top. Microbiol. Immunol. 78:171‐199.
  Nash, H.A. and Robertson, C.A. 1981. Purification and properties of the Escherichia coli protein factor required for lambda integrative recombination. J. Biol. Chem. 256:9246‐9253.
  Ptashne, M. 1992. A Genetic Switch: Phage (Lamda) and Higher Organisms. Cell Press, Cambridge, MA.
  Reboul, J., Vaglio, P., Tzellas, N., Thierry‐Mieg, N., Moore, T., Jackson, C., Shin‐I, T., Kohara, Y., Thierry‐Mieg, D., Thierry‐Mieg, J., Lee, H., Hitti, J., Doucette‐Stamm, L., Hartley, J.L., Temple, G.F., Brasch, M.A., Vandenhaute, J., Lamesch, P.E., Hill, D.E., and Vidal, M. 2001. Open‐reading‐frame sequence tags (OSTs) support the existence of at least 17,300 genes in C. elegans. Nat. Genet. 27:332‐336.
  Reboul, J., Vaglio, P., Rual, J.F., Lamesch, P., Martinez, M., Armstrong, C.M., Li, S., Jacotot, L., Bertin, N., Janky, R., Moore, T., Hudson, J.R. Jr, Hartley, J.L., Brasch, M.A., Vandenhaute, J., Boulton, S., Endress, G.A., Jenna, S., Chevet, E., Papasotiropoulos, V., Tolias, P.P., Ptacek, J., Snyder, M., Huang, R., Chance, M.R., Lee, H., Doucette‐Stamm, L., Hill, D.E., and Vidal, M. 2003. C. elegans ORFeome version 1.1: Experimental verification of the genome annotation and resource for proteome‐scale protein expression. Nat. Genet. 34:35‐41.
  Rolfs, A., Montor, W.R., Yoon, S.S., Hu, Y., Bhullar, B., Kelley, F., McCarron, S., Jepson, D.A., Shen, B., Taycher, E., Mohr, S.E., Zuo, D., Williamson, J., Mekalanos, J., and LaBaer, J. 2008a. Production and sequence validation of a complete full length ORF collection for the pathogenic bacterium Vibrio cholerae. Proc. Natl. Acad. Sci. U.S.A. 105:4364‐4369.
  Rolfs, A., Hu, Y., Ebert, L., Hoffman, D., Zuo, D., Ramachandran, N., Raphael, J., Kelley, F., McCarron, S., Jepson, D.A., Shen, B., Baqui, M.M.A., Pearlberg, J., Taycher, E., DeLoughery, C., Hoerlein, A., Korn, B., and LaBaer, J. 2008b. A biomedically enriched collection of 7000 human ORF clones. PLoS One. 3:e1528.
  SantaLucia, J. 1998. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest‐neighbor thermodynamics. Proc. Natl. Acad. Sci. U.S.A. 95:1460‐1465.
  Saul, J., Petritis, B., Sau, S., Rauf, F., Gaskin, M., Ober‐Reynolds, B., Mineyev, I., Magee, M., Chaput, J., Qiu, J., and LaBaer, J. 2014. Development of full‐length human protein production pipeline. Protein Sci. 23:1123‐1135.
  Thanawastien, A., Montor, W.R., LaBaer, J., Mekalanos, J.J., and Yoon, S.S. 2009. Vibrio cholerae proteome‐wide screen for immunostimulatory proteins identifies phosphatidylserine decarboxylase as a novel toll‐like receptor 4 agonist. PLoS Path. 5:e1000556.
  Walhout, A.J., Temple, G.F., Brasch, M.A., Hartley, J.L., Lorson, M.A, van den Heuvel, S., and Vidal, M. 2000. GATEWAY recombinational cloning: Application to the cloning of large numbers of open reading frames or ORFeomes. Methods Enzymol. 328:575‐592.
  Weisberg, R.A. and Landy, A. 1983. Site‐specific recombination in phage lamda. In Lamda II (R.A. Weisberg, ed.), pp. 211‐250. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  Yamada, K., Lim, J., Dale, J.M., Chen, H., Shinn, P., Palm, C.J., Southwick, A.M., Wu, H.C., Kim, C., Nguyen, M., Pham, P., Cheuk, R., Karlin‐Newmann, G., Liu, S.X., Lam, B., Sakano, H., Wu, T., Yu, G., Miranda, M., Quach, H.L., Tripp, M., Chang, C.H., Lee, J.M., Toriumi, M., Chan, M.M., Tang, C.C., Onodera, C.S., Deng, J.M., Akiyama, K., Ansari, Y., Arakawa, T., Banh, J., Banno, F., Bowser, L., Brooks, S., Carninci, P., Chao, Q., Choy, N., Enju, A., Goldsmith, A.D., Gurjal, M., Hansen, N.F., Hayashizaki, Y., Johnson‐Hopson, C., Hsuan, V.W., Iida, K., Karnes, M., Khan, S., Koesema, E., Ishida, J., Jiang, P.X., Jones, T., Kawai, J., Kamiya, A., Meyers, C., Nakajima, M., Narusaka, M., Seki, M., Sakurai, T., Satou, M., Tamse, R., Vaysberg, M., Wallender, E.K., Wong, C., Yamamura, Y., Yuan, S., Shinozaki, K., Davis, R.W., Theologis, A., and Ecker, J.R. 2003. Empirical analysis of transcriptional activity in the Arabidopsis genome. Science 302:842‐846.
  Yu, X., Bian, X., Throop, A., Song, L., Del Moral, L., Park, J., Seiler, C., Fiacco, M., Steel, J., Hunter, P., Saul, J., Wang, J., Qiu, J., Pipas, J.M., and LaBaer, J. 2014. Exploration of Panviral proteome: High‐throughput cloning and functional implications in virus‐host interactions. Theranostics 8:808‐822.
  Zhu, B., Cai, G., Hall, E.O., and Freeman, G.J. 2007. In‐Fusion assembly: Seamless engineering of multidomain fusion proteins, modular vectors, and mutations. BioTechniques 43:34‐359.
Internet Resources
  http://www.lifetechnologies.com/us/en/home/life‐science/cloning/gateway‐cloning.html
  Highly recommended website for information and troubleshooting related to Life Technologies Gateway Technology.
  http://www.clontech.com/US/Products/Cloning_and_Competent_Cells/Cloning_Kits/Cloning_Kits‐HD‐Liquid
  Highly recommended website for information and troubleshooting related to Clontech In‐Fusion Technology.
  https://www.neb.com/products/e5510‐gibson‐assembly‐cloning‐kit
  Highly recommended website for information related to New England Biolabs Gibson Assembly Cloning kit.
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