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Revision as of 04:21, 25 August 2021



pTol2: a Tol2 vector - the Tol2 transposable element

要顯示的連結文字或圖片放這裡


T--Mingdao--BBa K3728002-1.png

ThisTol2 transposon system is highly used in zebrafish transgenesis. The transposase protein (TPase) is from the Medaka fish (Oryzias latipes) aka Japanese rice fish, which catalyzes the transposition of the Tol2 elements through cut-and-paste mechanism. The minimal transposable Tol2 sequence (mTol2) contains 200-bp left arm and 150-bp right arm. Up to 11kb DNA insert between Tol2 sequence can be integrated into the genome of nearly all vertebrates including zebrafish, frog, chicken, mouse, and human [1].

ThisA further application in synthetic biology was demonstrated by Jun Ni, et. al.[2], in which the recombinant TPase protein is fully functional in HeLa cell line and Zebrafish germline cells. In addition, the TPase can be expressed under T7 promoter in E. coli BL21 and purified with N-terminal 6xHis tag. The transposase is active in vitro and mediated the integration of DNA fragments between plasmids with Tol2 elements.

ThisIn our study, we constructed BioBrick Parts of T7-TPase (BBa_K3728001) and the BioBrick compatible Tol2 vectors (BBa_K3728003) with reporter (KanR:BBa_K3728004; GFP:BBa_K3728005; RFP:BBa_K3728006; amilCP:BBa_K3728007) and Phi29 DNA polymerase genes (BBa_K3728008). We prepared the In vitro transcription-translation (TXTL) system [3][4]and expressed the functional reporter proteins. The recombinant TPase and Phil29 DNA polymerase with His tag were expressed in E. coli BL21. The purified proteins were functional in the plasmid integration assay and rolling circle amplification(RCA) application, respectively.


CONSTRUCTION – BIOBRICK COMPATIBLE VECTOR

CHARACTERIZATION – PLASMID INTEGRATION

APPLICATION – PHAGE ENGINEERING

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 3452
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 29
    Illegal NotI site found at 3458
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 3452
    Illegal BglII site found at 3340
    Illegal XhoI site found at 3419
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found at 3452
    Illegal suffix found at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found at 3452
    Plasmid lacks a suffix.
    Illegal XbaI site found at 3467
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NgoMIV site found at 642
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI site found at 1818
    Illegal SapI.rc site found at 2900


REFERENCE

  1. ↑ Kawakami K. Tol2: a versatile gene transfer vector in vertebrates. Genome Biol. 2007;8 Suppl 1(Suppl 1):S7. doi: 10.1186/gb-2007-8-s1-s7
  2. ↑ Ni J, Wangensteen KJ, Nelsen D, Balciunas D, Skuster KJ, Urban MD, Ekker SC. Active recombinant Tol2 transposase for gene transfer and gene discovery applications. Mob DNA. 2016 Mar 31;7:6. doi: 10.1186/s13100-016-0062-z.
  3. ↑ Garenne D, Noireaux V. Cell-free transcription-translation: engineering biology from the nanometer to the millimeter scale. Curr Opin Biotechnol. 2019 Aug;58:19-27. doi: 10.1016/j.copbio.2018.10.007.
  4. ↑ Rustad M, Eastlund A, Marshall R, Jardine P, Noireaux V. Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System. J Vis Exp. 2017 Aug 17;(126):56144. doi: 10.3791/56144.