Part:BBa_K1993011

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:

   
   

   
   
   

   Figure2. Expression of dTomato in 293FT cells.
   
   
   
   
   

   Figure 3. 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