Part:BBa_K3728000
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
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 DNA fragment was cloned into pSB1C3 as a basic BioBrick part (TPase/pSB1C3, <a href= “https://parts.igem.org/Part:BBa_K3376000”>Part:BBa_K3376000</a>). 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
- ↑ 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.
- ↑ 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
- ↑ 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1554
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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