Difference between revisions of "Part:BBa K1993011"
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'''Figure 3. Expression of dTomato in MSCs.'''<br>
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Revision as of 08:39, 17 October 2016


CXCR4-IRES-Luciferase-T2A-dtomato-T2A-hFTH

Functions:

With the purpose of increasing homing efficiency of MSCs and illustrating their distribution, we constructed a plasmid BBa_K1993008 under the control of EF-1α. (Figure 1) After that, plasmid BBa_K1993008 would be transducted into MSCs by lentivirus expression vector. What’s more, we confirmed its function in vitro and in vivo (DTH).

Figure 1 EF-1α-CXCR4-Luciferase-T2A-dTomato-T2A-hFTH

Details:

  1. Elongation factor-1α (EF-1α), a constitutive promoter, is responsible for mediating the recruitment of aminoacyl-tRNA to the A-site of the 80S ribosome during protein synthesis.
  2. CXCR4 is a chemokine receptor and its corresponding ligand is CXCL12. (Details could be seen on [xxxxxxxxxxx]
  3. Internal ribosome entry site (IRES) is an RNA element that allows for translation initiation in an end-independent manner, as part of the greater process of protein synthesis. (Details could be seen on [parts.igem.org/Part:BBa_K1993016 BBa_K1993016])
  4. Luciferase: Firefly (Photinus pyralis) Luciferase (Details can be seen from [parts.igem.org/Part:BBa_K1993018 BBa_K1993018]) is a kind of oxidative enzyme that produce bioluminescence. It is able to oxidize luciferin and produce detectable bioluminescence. It’s convenient to observe biological processes, especially allowing for non-invasive observation of cells.
  5. T2A： Between every two protein coding sequence, we added T2A a good candidate to replace IRES because of its small size and high cleavage efficiency between genes upstream and downstream of the 2A peptide. [1](Details can be seen from [parts.igem.org/Part:BBa_K1993019 BBa_K1993019]) between every two protein coding sequences to ensure all the coding sequences express as expected. (Figure 1)
  6. dTomato (Details can be seen from [parts.igem.org/Part:BBa_K1993020 BBa_K1993020]) is a kind of red fluorescent protein used for observation in vitro conveniently and easily;
  7. hFTH (See details in [parts.igem.org/Part:BBa_K1993021 BBa_K1993021]), another protein that could be observed in human body by MRI. (Figure 1) What’s more, in order to make sure the expression of all three proteins, we added a T2A part (Details can be seen from [parts.igem.org/Part:BBa_K1993019 BBa_K1993019]) between every two protein coding sequences. (Figure 1)

    Results:




    1. Expression of dTomato in 293FT cells.




    2. Expression of dTomato in MSCs.



    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 494
    • 23
      COMPATIBLE WITH RFC[23]
    • 25
      COMPATIBLE WITH RFC[25]
    • 1000
      INCOMPATIBLE WITH RFC[1000]
      Illegal SapI.rc site found at 2496