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

It is impractical for most synthetic biologists to synthesize more than several kilobases of completely synthetic DNA. It is often desirable to build bigger pieces at lower costs and faster speeds than de novo synthesis is currently able to accomplish.

Over the years, many different strategies have been developed to assemble DNA in flexible ways that suit different purposes. These strategies typically employ purification of enzymes that are known to modify DNA in specific ways and these methods of action can be exploited for designing and building specific sequences of DNA. For example, restriction enzymes are used to cut DNA in a specific manner upon recognition of a specific nucleotide sequence. Polymerases and endonucleases add or remove nucleotides to make double stranded DNA from a single stranded template or to create single stranded DNA from double stranded DNA. Many of the modern techniques take advantage of recombination machinery that break DNA from one location to reattach it to another location. In other cases, the endogenous enzymes in a host are utilized to manipulate DNA without the need for prior purification. For example, in some methods endogenous DNA ligase is used to repair single stranded breaks (known as "nicks") to complete the formation of fully circular DNA.

Ultimately, these methods generally require transformation into a host where endogenous enzymes are used to complete the genetic manipulation and replicate (clone) the genetic material. This allows for the expression of the desired proteins to test the ability of the engineered system or for the purification of the genetic material itself such that it can be used for further manipulation, study, or storage.

While there are many specific protocols for the numerous methods of cloning, most share reasonable overlap in their underlying mechanisms of action. Broadly speaking, methods may rely primarily on restriction enzymes, polymerase chain reaction (PCR), or on homologous recombination.

Restriction Enzyme

Restriction enzymes recognize a specific nucleotide sequence and then cut the DNA in such a way that results in a double stranded break. If the enzyme cuts within the recognition site, it is classified as a Type I restriction enzyme. If the enzyme cuts outside of the restriction site, the enzyme is classified as a Type II restriction enzyme. Restriction enzymes can also be classified by if the DNA break results in a straight cut resulting in a blunt end or with a jagged cut resulting in a sticky end.

For example: EcoRI digestion produces "sticky" ends,

http://en.wikipedia.org/wiki/File:EcoRI_restriction_enzyme_recognition_site.svg

whereas SmaI restriction enzyme cleavage produces "blunt" ends:

http://en.wikipedia.org/wiki/File:SmaI_restriction_enzyme_recognition_site.svg

The Registry of Biological Parts has developed [The_BioBricks_Foundation:Standards/Technical/Formats | standards] for the type and position of restriction enzyme sites to be used when building DNA using restriction enzymes in order to ensure compatibility in the connecting of biological parts. Two of the more popular standards are named and listed below:

  • BioBricks
  • BglBricks

One modification to the standard restriction enzyme method is the use of an enzyme that recognizes a non-palindromic sequence. This results in the ability to cut with a single restriction enzyme and still maintain directionality of the biological part.

  • CpoI directional cloning
  • golden gate
  • MoClo [5]
  • GoldenBraid

Polymerase Chain Reaction

Ligation Independent Cloning (LIC) [6]
TOPO TA Cloning (Invitrogen) [7][8]
Splice by Overlap Extension (SOE) PCR [9]
Polymerase Cycling Assembly (PCA) [10]

Recombination/Homology

  • In-Fusion (Clontech) poxvirus DNA polymerase with 3′–5′ exonuclease activity [11][12]
  • In-Fusion BioBrick Assembly [13]
  • cold fusion (SBI)
  • 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) [14]
-Recombineering lambda red

More cloning strategies found here

Links of Interest

References

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

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

  7. Holton TA and Graham MW. . pmid:2020554. PubMed HubMed [Holton1991]
    TA cloning

  8. Shuman S. . pmid:1658796. PubMed HubMed [Shuman1991]
    TOPO

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

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

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

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

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

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

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

    [Hillson2012]

  16. Tian J, Gong H, Sheng N, Zhou X, Gulari E, Gao X, and Church G. . pmid:15616567. PubMed HubMed [Tian2004]
  17. 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]
  18. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, and Smith HO. . pmid:19363495. PubMed HubMed [Gibson2009]
    oligonucleotide assembly in vitro

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

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

  22. 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]
  23. Hughes RA, Miklos AE, and Ellington AD. . pmid:21601682. PubMed HubMed [Hughes2011]
    Gene Synthesis Review

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

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

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

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

    //T5 exonuclease recombination

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

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

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

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

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

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

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

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

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

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

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

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

All Medline abstracts: PubMed HubMed
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