Recursive Directional Ligation Approach for Cloning Recombinant Spider Silks

  • Nina Dinjaski
  • Wenwen Huang
  • David L. Kaplan
Part of the Methods in Molecular Biology book series (MIMB, volume 1777)


Recent advances in genetic engineering have provided a route to produce various types of recombinant spider silks. Different cloning strategies have been applied to achieve this goal (e.g., concatemerization, step-by-step ligation, recursive directional ligation). Here we describe recursive directional ligation as an approach that allows for facile modularity and control over the size of the genetic cassettes. This approach is based on sequential ligation of genetic cassettes (monomers) where the junctions between them are formed without interrupting key gene sequences with additional base pairs.

Key words

Cloning Recombinant DNA techniques Recursive ligation Spider silk 



We thank the NIH (P41 EB002520), the AFOSR, and the NSF for support of this work.


  1. 1.
    Yigit S, Dinjaski N, Kaplan DL (2016) Fibrous proteins: at the crossroads of genetic engineering and biotechnological applications. Biotechnol Bioeng 113:913–929CrossRefGoogle Scholar
  2. 2.
    Lin S, Ryu S, Tokareva O, Gronau G, Jacobsen MM, Huang W, Rizzo DJ, Li D, Staii C, Pugno NM, Wong JY, Kaplan DL, Buehler MJ (2015) Predictive modelling-based design and experiments for synthesis and spinning of bioinspired silk fibres. Nat Commun 6:6892CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Huang W, Krishnaji ST, Hu X, Kaplan D, Cebe P (2011) Heat capacity of spider silk-like block copolymers. Macromolecules 44:5299–5309CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Krishnaji ST, Bratzel G, Kinahan ME, Kluge JA, Staii C, Wong JY, Buehler MJ, Kaplan DL (2013) Sequence-structure-property relationships of recombinant spider silk proteins: integration of biopolymer design, processing, and modeling. Adv Funct Mater 23:241–253CrossRefGoogle Scholar
  5. 5.
    Huang W, Krishnaji S, Kaplan D, Cebe P (2012) Thermal analysis of spider silk inspired di-block copolymers in the glass transition region by TMDSC. J Therm Anal Calorim 109:1193–1201CrossRefGoogle Scholar
  6. 6.
    Ittah S, Cohen S, Garty S, Cohn D, Gat U (2006) An essential role for the C-terminal domain of a dragline spider silk protein in directing fiber formation. Biomacromolecules 7:1790–1795CrossRefGoogle Scholar
  7. 7.
    Rabotyagova OS, Cebe P, Kaplan DL (2009) Self-assembly of genetically engineered spider silk block copolymers. Biomacromolecules 10:229–236CrossRefPubMedGoogle Scholar
  8. 8.
    Tokareva O, Michalczechen-Lacerda VA, Rech EL, Kaplan DL (2013) Recombinant DNA production of spider silk proteins. Microb Biotechnol 6:651–663CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Meyer DE, Chilkoti A (2002) Genetically encoded synthesis of protein-based polymers with precisely specified molecular weight and sequence by recursive directional ligation: examples from the elastin-like polypeptide system. Biomacromolecules 3:357–367CrossRefPubMedGoogle Scholar
  10. 10.
    Wang Q, Xia X, Huang W, Lin Y, Xu Q, Kaplan DL (2014) High throughput screening of dynamic silk-elastin-like protein biomaterials. Adv Funct Mater 24:4303–4310CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Wright ER, Conticello VP (2002) Self-assembly of block copolymers derived from elastin-mimetic polypeptide sequences. Adv Drug Deliv Rev 54:1057–1073CrossRefPubMedGoogle Scholar
  12. 12.
    Prince JT, McGrath KP, DiGirolamo CM, Kaplan DL (1995) Construction, cloning, and expression of synthetic genes encoding spider dragline silk. Biochemistry 34:10879–10885CrossRefPubMedGoogle Scholar
  13. 13.
    Higashiya S, Topilina NI, Ngo SC, Zagorevskii D, Welch JT (2007) Design and preparation of β-sheet forming repetitive and block-copolymerized polypeptides. Biomacromolecules 8:1487–1497CrossRefPubMedGoogle Scholar
  14. 14.
    Huang W, Krishnaji ST, Tokareva OR, Kaplan D, Cebe P (2014) Influence of water on protein transitions: morphology and secondary structure. Macromolecules 47:8107–8114CrossRefGoogle Scholar
  15. 15.
    Krishnaji ST, Huang W, Rabotyagova O, Kharlampieva E, Choi I, Tsukruk VV, Naik R, Cebe P, Kaplan DL (2011) Thin film assembly of spider silk-like block copolymers. Langmuir 27:1000–1008CrossRefPubMedGoogle Scholar
  16. 16.
    Huang W, Krishnaji S, Rabotyagova Tokareva O, Kaplan D, Cebe P (2014) Influence of water on protein transitions: thermal analysis. Macromolecules 47:8098–8106CrossRefGoogle Scholar
  17. 17.
    Xia X-X, Qian Z-G, Ki CS, Park YH, Kaplan DL, Lee SY (2010) Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber. Proc Natl Acad Sci 107:14059–14063CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Sambrook J, Russell DW (2001) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Biomedical EngineeringTufts UniversityMedfordUSA

Personalised recommendations