This summer, we wanted to construct a dual-luciferase miRNA target expression plasmid and perform a dual luciferase assay, to evaluate the microRNA (miRNA) binding ability of miRNA binding sequences.
MiR-214 and its target sequence RNF8 were chosen in our project, unfortunately, the 3’ untranslated region of RNF8 is not compatible with BioBrick RFC as multiple illegal sites were contained in it. This troubles us a lot and caused a lot of difficulties in our experiment.
Thus, we decided to do something to improve the part BBa_I712019, to optimize its function in a dual-luciferase miRNA target expression system. Two restriction sites of BsmBI were added to the 3’ end of firefly luciferase, through which, the 3’ untranslated region of any gene can be inserted using Golden-Gate Assembly, with no worry about the multiple illegal sites contained in it.
We believe that this improvement simplifies the construction of a dual-luciferase miRNA target expression plasmid and greatly facilitates the related miRNA research.
Figure 1. Schematic representation of the workflow of the plasmid construction. (a) The improvement of the part. (b) The restriction sites of BsmBI. (c) The construction of the plasmid.