<b>Bioprospecting Novel Luciferase Genes from Museum Coleoptera</b>

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.鞘翅目（Coleoptera）博物馆馆藏标本蕴含具备生物技术应用潜力的遗传物质，非破坏性DNA提取技术可在保留重要标本完整性的前提下，同时获取其线粒体DNA与基因组DNA。针对线粒体细胞色素C氧化酶亚基I（MT-COI）基因区域的微型条形码技术，可帮助识别并排除已知物种，使其不进入后续研究流程。本研究使用靶向荧光素酶基因（luciferase gene）第四外显子、内含子与第五外显子区域的简并杂交寡核苷酸共识（Consensus Degenerate Hybrid Oligonucleotide, CODEHOP）引物，对样本中的荧光素酶基因序列进行检测，并剔除携带已知荧光素酶序列的样本。源自萤火虫（Photinus pyralis）的生物素标记荧光素酶基因探针，可用于富集潜在的荧光素酶基因片段，以供下一代测序（next-generation sequencing）使用。随后通过生物信息学分析套件（bioinformatic analysis suite），从2012年在哥斯达黎加采集的此前未被鉴定的萤火虫样本中，成功识别出一条荧光素酶基因序列。本研究证实该新发现的荧光素酶（命名为CRLuc）可催化生物发光反应，并测定了其发射光谱、底物ATP与D-荧光素的米氏常数（Km），以及该酶的pH稳定性。 本次上传的文件对应以下文件夹： 【生物发光活性筛选-大肠杆菌（E. coli）】板块包含一个初筛文件夹，其内包含"Costa Rica Luc gBlock 100ul spread plate DLH2"相关的.bvr、.cri、.trp及.pxl格式文件，此类文件可通过M3Vision软件打开。另有一个复筛文件夹，其内包含不同波长的采集数据，同样可通过M3Vision软件进行分析，波长涵盖455、480、505、530、555、580、605、630、655、680、705、730、755、780，以及Open1和Open2。 另有一份Word文档，包含简并杂交寡核苷酸共识（CODEHOP）引物的Sanger测序（Sanger Sequencing）所需的DNA序列。 【动力学约束测定-ATP】板块包含CRLuc文件夹与Ppy文件夹：CRLuc文件夹内为ATP浓度范围0.1μM至1000μM的相关Excel文件及ATP反应曲线图；Ppy文件夹内同样包含ATP浓度范围0.1μM至1000μM的相关Excel文件及ATP反应曲线图。 【动力学约束测定-DLH2】板块包含CRLuc文件夹与Ppy文件夹：CRLuc文件夹内为D-荧光素浓度范围0.1μM至200μM的相关Excel文件及DLH2反应曲线图；Ppy文件夹内同样包含D-荧光素浓度范围0.1μM至200μM的相关Excel文件及DLH2反应曲线图。 【活性pH依赖性测定】板块包含CRLuc文件夹与Ppy文件夹：CRLuc文件夹内为pH值范围6.3至8.8的相关Excel文件及pH依赖性活性曲线图；Ppy文件夹内同样包含pH值范围6.3至8.8的相关Excel文件及pH依赖性活性曲线图。 【生物发光光谱pH依赖性测定】板块包含CRLuc文件夹与Ppy文件夹：CRLuc文件夹内为pH值6.3至8.8的生物发光光谱数据相关Excel文件及对应曲线图；Ppy文件夹内同样包含pH值6.3至8.8的生物发光光谱数据相关Excel文件及对应曲线图。 【富集qPCR数据】文件夹包含两份Excel文件，分别为"All LIBRARIES pre & post enrich"与"Lnoc & Ppy PrePost enrich"。 【高分辨率光谱】文件夹包含两份Excel文件，分别为"CRLuc High Resolution Spectra"与"Ppy High Resolution Spectra"。