Bioprospecting Novel Luciferase Genes from Museum Coleoptera

Museum collections of Coleoptera contain genetic material of potential interest to biotechnology, and non-destructive DNA extraction enables the preservation of important specimens with concomitant release of mitochondrial and genomic DNA. Mini-barcoding of regions of the mitochondrial cytochrome oxidase subunit I (MT-COI) gene helps identify and eliminate known species from further investigation. Consensus Degenerate Hybrid Oligonucleotide (CODEHOP) primers targeting the region of the luciferase gene spanning the fourth exon, intron, and fifth exon were used to detect luciferase gene content and eliminate samples containing known luciferase sequences. Biotinylated luciferase gene probes from the firefly \textit{Photinus pyralis} enabled the enrichment of potential luciferase gene fragments for next-generation sequencing. A bioinformatic analysis suite was then used to identify a luciferase gene sequence from a previously unidentified firefly originally collected in Costa Rica in 2012. We demonstrate that this newly discovered luciferase, termed CRLuc, catalyses a bioluminescent reaction and we determined its emission spectra, Km for the substrates ATP and D-luciferin, and pH stability.

The files uploaded relate to the following folders:

Bioluminescence Activity Screening - E. coli has a Primary Screen folder containing 'Costa Rica Luc gBlock 100ul spread plate DLH2 .bvr, .cri, .trp and .pxl files which can be opened with M3Vision software. There is also a Secondary Screen folder which contains different wavelength acquisitions again analysable using M3Vision; 455, 480, 505, 530, 555, 580, 605, 630, 655, 680, 705, 730, 755, 780, Open1 and open2.

A word file contains DNA sequences for CODEHOP Sanger Sequencing.

Determination of Kinetic Constraints - ATP has a CRLuc folder containing Excel files with ATP concentrations from 0.1uM to 1000uM and ATP plots. The Ppy folder contains Excel files with ATP concentrations from 0.1uM to 1000uM and ATP plots.

Determination of Kinetic Constraints - DLH2 has a CRLuc folder containing Excel files with D-luciferin concentrations from 0.1uM to 200uM and DLH2 plots. The Ppy folder contains Excel files with DLH2 concentrations from 0.1uM to 200uM and DLH2 plots.

Determination of pH Dependence of Activities has a CRLuc folder containing Excel files with pH values from 6.3 to 8.8 and pH Dependence Activity Plots. The Ppy folder contains Excel files with pH values from 6.3 to 8.8 and pH Dependence Activity Plots.

Determination of pH Dependence of Bioluminescent Spectra has a CRLuc folder containing Excel files with pH bioluminescence spectra data from pH 6.3 to 8.8 and Plots. The Ppy folder contains Excel files with pH bioluminescence spectra data from pH 6.3 to 8.8 and Plots.

Enrichment qPCR Data folder contains two Excel files; All LIBRARIES pre & post enrich, and Lnoc & Ppy PrePost enrich.

High Resolution Spectra folder contains two Excel files; CRLuc High Resolution Spectra and Ppy High Resolution Spectra.