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Revision as of 04:12, 25 August 2021
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__NOTOC__
BBa_K3728002 short
[[File:T--Mingdao--BBa K3728002-1.png|600px|right]]
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 [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].
ThisA further application in synthetic biology was demonstrated by Jun Ni, et. al.[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.], 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 ([https://parts.igem.org/Part:BBa_K3728001 BBa_K3728001]) and the BioBrick compatible Tol2 vectors ([https://parts.igem.org/Part:BBa_K3728003 BBa_K3728003]) with reporter (KanR:[https://parts.igem.org/Part:BBa_K3728004 BBa_K3728004]; GFP:[https://parts.igem.org/Part:BBa_K3728005 BBa_K3728005]; RFP:[https://parts.igem.org/Part:BBa_K3728006 BBa_K3728006]; amilCP:[https://parts.igem.org/Part:BBa_K3728007 BBa_K3728007]) and Phi29 DNA polymerase genes ([https://parts.igem.org/Part:BBa_K3728008 BBa_K3728008]). We prepared the In vitro transcription-translation (TXTL) system [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.][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.]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===
BBa_K3728002 SequenceAndFeatures
=== REFERENCE ===
