Difference between revisions of "Part:BBa K3728000"
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=== CONSTRUCTION – TPase/pSB1C3 ===
 
=== CONSTRUCTION – TPase/pSB1C3 ===
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Oryzias latipes Tol2 transposase (TPase) gene sequence was taken from UniProt database ([https://www.uniprot.org/uniprot/Q9PVN3 UniProtKB - Q9PVN3]) and optimized based on E. coli codon preference. The TPase gene was designed with 6xHis tag and a GS linker at N-terminus and synthesized by Integrated DNA Technologies, Inc. (IDT). The part was checked by colony PCR and restriction enzymes and further confirmed by sequencing (Fig. 1).
 
Oryzias latipes Tol2 transposase (TPase) gene sequence was taken from UniProt database ([https://www.uniprot.org/uniprot/Q9PVN3 UniProtKB - Q9PVN3]) and optimized based on E. coli codon preference. The TPase gene was designed with 6xHis tag and a GS linker at N-terminus and synthesized by Integrated DNA Technologies, Inc. (IDT). The part was checked by colony PCR and restriction enzymes and further confirmed by sequencing (Fig. 1).
  

Revision as of 09:24, 17 October 2021


Tol2 transposase (Oryzias latipes) - TPase

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[1]. 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 [2].

ThisA further application in synthetic biology was demonstrated by Jun Ni, et. al.[3], 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.

CONSTRUCTION – TPase/pSB1C3

T--Mingdao--000photo1 1017.png

Oryzias latipes Tol2 transposase (TPase) gene sequence was taken from UniProt database (UniProtKB - Q9PVN3) and optimized based on E. coli codon preference. The TPase gene was designed with 6xHis tag and a GS linker at N-terminus and synthesized by Integrated DNA Technologies, Inc. (IDT). The part was checked by colony PCR and restriction enzymes and further confirmed by sequencing (Fig. 1).


PROTEIN EXPRESSION, PURIFICATION & ANALYSIS

IN VITRO INTEGRATION ASSAY

PHAGE ENGINEERING

APPLICATION - SALMONELLA DETECTION

THE LIMIT OF SALMONELLA PHAGE REPORTER

COMPARISON WITH CURRENT METHODS



Reference

  1. ↑ Urasaki A, Morvan G, Kawakami K. Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition. Genetics. 2006 Oct;174(2):639-49. doi: 10.1534/genetics.106.060244.
  2. ↑ 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
  3. ↑ 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.

Note: The map was generated and sponsored by SnapGene.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1554
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]