Part:BBa_K1993025
Luciferase-IRES-eGFP
Driven by our insights about realizing locating MSCs both in vivo and in vitro, we constructed a composite part expressing Luciferase and eGFP. Luciferase(Details can be seen from [parts.igem.org/Part:BBa_K1993015 BBa_K1993015]) is a generic term for the class of oxidative enzymes that produce bioluminescence by oxidizing luciferin substrate and responsible for regulating light production in a variety of organs. It’s convenient to observe biological processes, especially allowance for observation of cells non-invasively and specifically. In this composite part, we chose the gene of Renilla-luciferin 2-monooxygenase (RLuc) of 936 bp. Unlike Firefly Luciferase required ATP to catalyze, RLuc is a shorter version cooperating with O2 to oxidize luciferin. In consideration of testifying the location of MSCs in multiple ways and provide future teams with optional and economic way to observe, we enhanced the basic part of RLuc with the gene of eGFP(Details can be seen from [parts.igem.org/Part:BBa_K1993017 BBa_K1993017]), which is a modified form of GFP frequently used as a reporter of expression. The observation of these two marking proteins ensures accurate locating of MSCs. To ensure coexpression of Luciferase and eGFP, we introduced Internal ribosome entry site (IRES) (Details can be seen from [parts.igem.org/Part:BBa_K1993016 BBa_K1993016])between them, an RNA element that allows for translation initiation in an end-independent manner, as part of the greater process of protein synthesis.(Figure 1)
In our experiments, we constructed a full plasmid by adding EF-1α-CXCR4-T2A at upstream of this composite part. We confirmed its expression by fluorescent microscope and IVIS spectrum system. The results are shown as follows(Figure1, Figure2, Figure3):
Figure 1 The constitution of composite part
Figure2 Expression of eGFP in 293FT cells.
Figure 3 Expression of eGFP in MSCs.
Figure 4 Oxidation of Luciferin in living mouse.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 228
Illegal BamHI site found at 771 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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