CH391L/S13/DnaAssembly

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DNA synthesis and molecular cloning are tools used by synthetic biologists to create the biological "parts" needed to design and engineer biological devices and systems.

Contents

Introduction

As described before, synthetic biology captures a diverse, multi-disciplinary field. No matter which definition(s) becomes accepted, the ability to make and manipulate DNA is a vital component to practicing synthetic biology.

A large number of parts have been made by the synthetic biology community. Many can be found as part of the Registry of Standard Biological Parts. These modular genetic components are designed to be easy to acquire and assemble to facilitate the building of more complex biological devices. To learn more about the Registry and the biological parts known as BioBricks™, see the entry for the iGEM Registry.

The Registry of Standard Biological Parts is an attempt to create an annotated and characterized repository of biological parts. It is motivated in part because synthetic biologists rely on the ability to make testable biological units. While the parts registry is a useful resource, it is not comprehensive. The ability to manipulate and create genetic material is a necessary skill for being a successful synthetic biologists. This page details how to create DNA from small (<60 nts) oligonucleotides to larger genes (~400 nts) to genome sized (~500 ,000 nts) biological units. Many of the methods found here are the basis for the construction of the registry itself.

DNA Synthesis

Oligonucleotide Synthesis

Oligonucleotides are chemically synthesized from DNA phosphoramidite monomers. Briefly, activated phosphoramidite monomers are added in the 3' to 5' direction using a cyclical activation and blocking chemistry to obtain a DNA polymer linked by phosphodiester bonds.

Image:CH391L_S12_Phosphoramidite.png

Chemical synthesis is currently limited to oligonucleotides of about 200 nt in length.

Gene Synthesis

Gene synthesis, or artificial gene synthesis, refers to the process of creating a nucleic acid template for a gene in vitro, without the requirement of a preexisting DNA template. Soon after the elucidation of the genetic code and the description of the central dogma of molecular biology, there arose a need to synthesize genes de novo in order to study their biological function both in the test tube and in model organisms. Chemical synthesis of DNA has grown from an expensive and time-consuming process into a viable commercial industry capable of high-throughput manufacture of almost any scale of custom DNA molecules in almost any context. This allows species-specific gene optimization, creation of genes from rare or dangerous sources, and combinatorial assembly of any DNA sequence that can be chemically synthesized, even including non-traditional bases. The most advanced applications of gene synthesis have been applied to the recent creation of completely synthetic minimal genomes in prokaryotes.

Despite nearly four decades of progress in gene synthesis technologies, most DNA sequences used in modern molecular biology are assembled in part or in whole from naturally occurring templates. However this limits the scope and applications to previously existing genes and the results of large-scale genomic surveys of novel genes from nature. Modern gene synthesis relies heavily on advancements in chemical DNA oligonucleotide synthesis, with the primary challenges being scale, cost, fidelity and the eventual assembly of complete gene products.

An extensive, but not comprehensive, directory of commercial gene synthesis providers can be found at Genespace.

Economics


History of Gene Synthesis

Gene synthesis predates the invention of restriction enzymes and molecular cloning techniques by several years. The first gene to be completely synthesized in vitro was a 77-nt alanine transfer RNA by the laboratory of Har Gobind Khorana in 1972 [1]. This was the result of nearly five years of work and resulted in a DNA template without promoter or transcriptional control sequences. The first peptide- and protein-producing synthetic genes were created in 1977 and 1979, respectively [2, 3]. Steady advancement has led to recent synthesis of complete gene clusters tens of thousands of nucleotides in length, and ultimately a bacterial genome approximately 1.1 million bases in length [4].

Longest Published Synthetic DNA [1]
Longest Published Synthetic DNA [1]

Molecular Cloning

The methods generally require transformation into a host where the endogenous enzymes are used to complete the genetic manipulation and replicate (clone) the genetic material.

Restriction Enzyme

Standard Restriction Enzyme Digest
CpoI directional cloning

BioBricks
BglBricks
list of BioBrick Foundation Standards

Polymerase Chain Reaction

Ligation Independent Cloning (LIC) [5]
TOPO TA Cloning (Invitrogen)
Splice by Overlap Extension (SOE) PCR [6]
Polymerase Cycling Assembly (PCA) [7]

Recombination/Homology

  • In-Fusion (Clontech) poxvirus DNA polymerase with 3′–5′ exonuclease activity [8][9]
  • In-Fusion BioBrick Assembly [10]
  • cold fusion (SBI)
  • golden gate
  • MoClo [11]
  • GoldenBraid
  • Cre/Lox P1 phage (Clontech)
  • att lambda (gateway)
  • CloneEZ kit (Genescript) [2], recombination around a linearized vector
  • GENEART Seamless Cloning (Life Technologies previously Invitrogen previously DoGene)
  • SLIC sequence and ligation independent cloning T4 DNA polymerase (exonuclease)
  • Gibson T5 exonuclease, Phusion polymerase, Taq ligase
  • CPEC circular polymerase extension cloning
  • SLiCE (Seamless Ligation Cloning Extract) in vitro homologous recombination

In Vivo

-MAGIC (bacterial mating) [12]
-Recombineering lambda red

More cloning strategies found here

Links of Interest

References

  1. Khorana HG, Agarwal KL, Büchi H, Caruthers MH, Gupta NK, Kleppe K, Kumar A, Otsuka E, RajBhandary UL, Van de Sande JH, Sgaramella V, Terao T, Weber H, and Yamada T. . pmid:4571075. PubMed HubMed [Khorana1972]
  2. Itakura K, Hirose T, Crea R, Riggs AD, Heyneker HL, Bolivar F, and Boyer HW. . pmid:412251. PubMed HubMed [Itakura1977]
  3. Goeddel DV, Kleid DG, Bolivar F, Heyneker HL, Yansura DG, Crea R, Hirose T, Kraszewski A, Itakura K, and Riggs AD. . pmid:85300. PubMed HubMed [Goeddell1979]
  4. Gibson DG, Glass JI, Lartigue C, Noskov VN, Chuang RY, Algire MA, Benders GA, Montague MG, Ma L, Moodie MM, Merryman C, Vashee S, Krishnakumar R, Assad-Garcia N, Andrews-Pfannkoch C, Denisova EA, Young L, Qi ZQ, Segall-Shapiro TH, Calvey CH, Parmar PP, Hutchison CA 3rd, Smith HO, and Venter JC. . pmid:20488990. PubMed HubMed [Gibson2010]
    genome replacement

  5. Aslanidis C and de Jong PJ. . pmid:2235490. PubMed HubMed [Aslanidis1990]
    LIC

  6. Higuchi R, Krummel B, and Saiki RK. . pmid:3045756. PubMed HubMed [Higuchi1988]
    SOE-PCR

  7. Stemmer WP, Crameri A, Ha KD, Brennan TM, and Heyneker HL. . pmid:7590320. PubMed HubMed [Stemmer1995]
    PCA

  8. Zhu B, Cai G, Hall EO, and Freeman GJ. . pmid:17907578. PubMed HubMed [Zhu2007]
    in-fusion

  9. Benoit RM, Wilhelm RN, Scherer-Becker D, and Ostermeier C. . pmid:16289702. PubMed HubMed [Benoit2006]
    in-fusion

  10. Sleight SC, Bartley BA, Lieviant JA, and Sauro HM. . pmid:20385581. PubMed HubMed [Sleight2010]
    In-Fusion biobrick

  11. Weber E, Engler C, Gruetzner R, Werner S, and Marillonnet S. . pmid:21364738. PubMed HubMed [Weber2011]
    MoClo

  12. Li MZ and Elledge SJ. . pmid:15731760. PubMed HubMed [Li2005]
    MAGIC, bacterial mating approach

  13. j5 DNA Assembly Design Automation Software doi: 10.1021/sb2000116

    [Hillson2012]

  14. Tian J, Gong H, Sheng N, Zhou X, Gulari E, Gao X, and Church G. . pmid:15616567. PubMed HubMed [Tian2004]
  15. Gibson DG, Benders GA, Andrews-Pfannkoch C, Denisova EA, Baden-Tillson H, Zaveri J, Stockwell TB, Brownley A, Thomas DW, Algire MA, Merryman C, Young L, Noskov VN, Glass JI, Venter JC, Hutchison CA 3rd, and Smith HO. . pmid:18218864. PubMed HubMed [Gibson2008]
  16. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, and Smith HO. . pmid:19363495. PubMed HubMed [Gibson2009]
    oligonucleotide assembly in vitro

  17. Gibson DG. . pmid:19745056. PubMed HubMed [Gibson2009b]
    oligonucleotide assembly in yeast

  18. Gibson DG, Smith HO, Hutchison CA 3rd, Venter JC, and Merryman C. . pmid:20935651. PubMed HubMed [Gibson2010b]
  19. Gibson DG. . pmid:21601685. PubMed HubMed [Gibson2011]
    MIE paper

  20. Dymond JS, Richardson SM, Coombes CE, Babatz T, Muller H, Annaluru N, Blake WJ, Schwerzmann JW, Dai J, Lindstrom DL, Boeke AC, Gottschling DE, Chandrasegaran S, Bader JS, and Boeke JD. . pmid:21918511. PubMed HubMed [Dymond2011]
  21. Hughes RA, Miklos AE, and Ellington AD. . pmid:21601682. PubMed HubMed [Hughes2011]
    Gene Synthesis Review

  22. Werner S, Engler C, Weber E, Gruetzner R, and Marillonnet S. . pmid:22126803. PubMed HubMed [Werner2012]
    MoClo

  23. Sarrion-Perdigones A, Falconi EE, Zandalinas SI, Juárez P, Fernández-del-Carmen A, Granell A, and Orzaez D. . pmid:21750718. PubMed HubMed [SarrionPerdigones2011]
    GoldenBraid

  24. Engler C, Kandzia R, and Marillonnet S. . pmid:18985154. PubMed HubMed [Engler2008]
    GoldenGate

  25. Quan J and Tian J. . pmid:19649325. PubMed HubMed [Quan2009]
    CPEC

    //T5 exonuclease recombination

  26. Li MZ and Elledge SJ. . pmid:17293868. PubMed HubMed [Li2007]
    SLIC

  27. Li MZ and Elledge SJ. . pmid:22328425. PubMed HubMed [Li2012]
    SLIC

  28. Geu-Flores F, Nour-Eldin HH, Nielsen MT, and Halkier BA. . pmid:17389646. PubMed HubMed [GeuFlores2007]
    USER

  29. Horton RM, Cai ZL, Ho SN, and Pease LR. . pmid:2357375. PubMed HubMed [Horton2009]
    SOEing

  30. Czar MJ, Anderson JC, Bader JS, and Peccoud J. . pmid:19111926. PubMed HubMed [Czar2009]
    review

  31. Aslanidis C, de Jong PJ, and Schmitz G. . pmid:7580902. PubMed HubMed [Aslanidis1994]
    LIC

  32. Li C and Evans RM. . pmid:9321675. PubMed HubMed [Li1997]
    LIC

  33. Angrand PO, Daigle N, van der Hoeven F, Schöler HR, and Stewart AF. . pmid:10446259. PubMed HubMed [Angrand1999]
    lambda Red recombinase

  34. Hartley JL, Temple GF, and Brasch MA. . pmid:11076863. PubMed HubMed [Hartley2000]
    Gateway lambda Int

  35. Khalil AM, Julius JA, and Bachant J. . pmid:17702758. PubMed HubMed [Khalil2007]
    Gateway lambda Cre

  36. Larionov V, Kouprina N, Graves J, Chen XN, Korenberg JR, and Resnick MA. . pmid:8552668. PubMed HubMed [Larionov1996]
    Transformation-associated recombination (TAR) cloning

  37. Zhang Y, Werling U, and Edelmann W. . pmid:22241772. PubMed HubMed [Zhang2012]
    SLiCe

  38. Holton TA and Graham MW. . pmid:2020554. PubMed HubMed [Holton1991]
All Medline abstracts: PubMed HubMed
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