reaction for the assay can be seen below in Figure 8, which was taken from Dual-Glo Promega’s protocol. Figure 8. Recombinant Firefly Luciferase assay reaction. A series of test vectors were constructed by Dr. Nakesha Smith designed to produce recombinant firefly luciferase based on the riboswitch system. First, the reporter gene, firefly luciferase, was inserted into an expression vector. The pRSF.1b expression vector from Novagen was chosen for a number of reasons. It is a high copy number plasmid, confers Kanamycin resistance, contains an RSF origin and has an inducible T7 promoter. The plasmid map of the pRSF.1b vector is shown in Figure 9. Figure 9. Plasmid map of the pRSF.1b vector. The gene for firefly luciferase was cloned into the expression vector from the fusion vector from Promega. This vector is shown in Figure 10 below. Figure 10. Plasmid map of the pSP-luc+NF fusion vector. The resulting vector was the pRSF-Luc plasmid. The riboswitch constructs for the chosen bacteria were cloned into the of the pRSF-Luc vector using a 5’ untranslated megaprimer-based polymerase chain reaction mutagenesis strategy (Tyagi, 2004).
reaction for the assay can be seen below in Figure 8, which was taken from Promega’s Dual-Glo protocol. Figure 8. Recombinant Firefly Luciferase assay reaction. A series of test vectors were constructed by Dr. Nakesha Smith designed to produce recombinant firefly luciferase based on the riboswitch system. First, the reporter gene, firefly luciferase, was inserted into an expression vector. The pRSF.1b expression vector from Novagen was chosen for a number of reasons. It is a high copy number plasmid, confers Kanamycin resistance, contains an RSF origin and has an inducible T7 promoter. The plasmid map of the pRSF.1b vector is shown in Figure 9. Figure 9. Plasmid map of the pRSF.1b vector. The gene for firefly luciferase was cloned into the expression vector from the fusion vector from Promega. This vector is shown in Figure 10 below. Figure 10. Plasmid map of the pSP-luc+NF fusion vector. The resulting vector was the pRSF-Luc plasmid. The riboswitch constructs for the chosen bacteria were cloned into the 5’ untranslated of the pRSF-Luc vector using a megaprimer-based polymerase chain reaction mutagenesis strategy (Tyagi, 2004).