Difference between revisions of "Part:BBa K4046620"

Latest revision as of 00:51, 22 October 2021

hUBC-Kozak-cLuc

Cypridina luciferase is an enzyme that plays an important role in bioluminescence. In order to do this, it cleaves its substrate, vargulin, which produces a luminescent molecule. cLuc that is expressed in cells can be secreted into the culturing media, which allows it to be used as a dynamic reporter for the concentration of the compound of interest. This reporter can be used as a luminescence-based constitutive reporter to quantitatively and non-invasively assess cell behavior.

To test the gene expression of hUBC-cLuc, we transfected a plasmid containing this construct into HEK293T cells, and cell media samples were tested for luciferase data after 2 days using vargulin. Overall, the results demonstrate that the cLuc gene behaves as expected, as it induces luminescence activity when acting on its vargulin substrate.

Figure 1: Luminescence assay results for cLuc gene with hUBC promoter

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 1018
Illegal BglII site found at 1077
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 <partinfo>BBa_K4046620 short</partinfo>
-This is a constitutive reporter consisting of the hUBC promoter followed by the gene coding for Cypridina luciferase (cLuc), an enzyme that plays an important role in bioluminescence. cLuc that is expressed in cells can be secreted into the culturing media, which allows it to be used as a dynamic reporter for the concentration of the compound of interest. In between these two sequences is the Kozak sequence, which provides a start point for eukaryotic translation. This construct was added to a pcDNA5 plasmid and was created as one of our constitutive reporters; it will be used as a construct into which we insert our <i>dhdO</i> binding sequences in order to build a dynamic reporter for the compound D-2-HG.
+Cypridina luciferase is an enzyme that plays an important role in bioluminescence. In order to do this, it cleaves its substrate, vargulin, which produces a luminescent molecule. cLuc that is expressed in cells can be secreted into the culturing media, which allows it to be used as a dynamic reporter for the concentration of the compound of interest. This reporter can be used as a luminescence-based constitutive reporter to quantitatively and non-invasively assess cell behavior.
+To test the gene expression of hUBC-cLuc, we transfected a plasmid containing this construct into HEK293T cells, and cell media samples were tested for luciferase data after 2 days using vargulin. Overall, the results demonstrate that the cLuc gene behaves as expected, as it induces luminescence activity when acting on its vargulin substrate.
+[[File:T--Duke--hUBC_curve.png|800 px]] <br>
+<b><font size="+1">Figure 1: Luminescence assay results for cLuc gene with hUBC promoter</font></b>