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
ThisOryzias 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).
Figure 1 | 6xHis-GS-TPase/pSB1C3 construct check. DNAs were run electrophoresis on 1% agarose gel with 1kb marker. (a) 4 colonies were subject to PCR with TPase-specific forward and reverse primers (PCR product size: ~2000 bp). (b) The DNAs were extracted and digested by EcoRI and BamHI (2893, 870 and 235 bps).
PROTEIN EXPRESSION, PURIFICATION & ANALYSIS
ThisHis-tagged Tol2 transposase (TPase) was assembled with a T7 promoter and expressed in a cell-free in vitro transcription-translation (TXTL) system using the bacterial cytoplasmic extracts prepared from IPTG-induced E. coli Rosetta 2(DE3) cells. The His-tagged proteins were further purified through Nickel column. The protein concentration was measured and analyzed on SDS-PAGE and Coomassie Blue Staining (Fig. 2). The protein was shown at around 70 kDa as the same size as the predicted TPase protein (664 a.a., 75 kDa). The Elution #4, #5 and #6 were collected and used for further studies.
Figure 2 | His-Tol2 transposase was expressed in TXTL and purified by Nickel column. 10 μg of protein lysates were analyzed by SDS-PAGE and Coomassie Blue Staining using 4–12% gradient gel (NuPAGE™, Thermo Fisher Scientific Inc.) Lane: (1) PageRuler™ Prestained Protein Ladder, (2) E. coli Rosetta 2(DE3) cell extracts (no DNA control), (3) total lysates in TXTL, (4) flow-through, (5) wash-through, (6) Elution #4, (7) Elution #5, (8) Elution #6, (9) Elution #7, (10) Elution #8, (11) Elution #9.
IN VITRO INTEGRATION ASSAY
ThisIn vitro integration assay was used by Jun Ni, et al. to characterize the activity of purified recombinant Tol2 transposase (TPase) and the transposition of Tol2 mobile element[4]. We prepared the purified TPase from TXTL (Fig. 2) and performed PCR to generate KanR, ldhp-GFP-Tr and ldhp-amilCP-Tr (expressing blue color) DNA fragments flanked by 200-bp right and 150-bp left arms of pTol2Part:BBa_K3728002. The mixtures of TPase, Tol2 mobile inserts and a target plasmid of pSB1C3 were incubated at 30°C for 2 hours. The resulting DNAs were cleaned up and subjected to transform E. coli DH5α competent cells. The colonies displaying kanamycin resistance, green fluorescence or blue color were counted as successful jumping to plasmids by active purified TPase. And the integration rate was calculated by comparing with chloramphenicol resistance or red colonies from pSB1C3 backbone carrying BBa_J04450 part (i.e., RFP coding device).
PHAGE ENGINEERING
APPLICATION - SALMONELLA DETECTION
THE LIMIT OF SALMONELLA PHAGE REPORTER
COMPARISON WITH CURRENT METHODS
USAGE: SAFETY ISSUE - GENE DRIVE
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.
- ↑ 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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