Part:BBa_K3728002

pTol2: a Tol2 vector - the Tol2 transposable element

Template:SpacesTol2 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 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)Template:123.

A further application in synthetic biology was demonstrated by Jun Ni, et. al. (2), in which the recombinant TPase is fully functional in vivo and characterized in HeLa cell line and Zebrafish germline assays. 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.

In our study, we constructed BioBrick Parts of T7-TPase and the BioBrick compatible Tol2 vectors with reporter genes. We prepared the In vitro transcription-translation (TXTL) system (3, 4) and expressed the functional recombinant TPase which was characterized by plasmid integration assay and further applied in phage engineering.

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.

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