Project description:Marine luciferases are regularly employed as useful reporter molecules across a range of various applications. However, attempts to transition expression from their native eukaryotic environment into a more economical prokaryotic, i.e. bacterial, expression system often presents several challenges. Specifically, bacterial protein expression inherently lacks chaperone proteins to aid in the folding process, while Escherichia coli presents a reducing cytoplasmic environment in. These conditions contribute to the inhibition of proper folding of cysteine-rich proteins, leading to incorrect tertiary structure and ultimately inactive and potentially insoluble protein. Vargula luciferase (Vluc) is a cysteine-rich marine luciferase that exhibits glow-type bioluminescence through a reaction between its unique native substrate and molecular oxygen. Because most other commonly used bioluminescent proteins exhibit flash-type emission kinetics, this emission characteristic of Vluc is desirable for high-throughput applications where stability of emission is required for the duration of data collection. A truncated form of Vluc that retains considerable bioluminescence activity (55%) compared to the native full-length protein has been reported in the literature. However, expression and purification of this luciferase from bacterial systems has proven difficult. Herein, we demonstrate the expression and purification of a truncated form of Vluc from E. coli. This truncated Vluc (tVluc) was subsequently characterized in terms of both its biophysical and bioluminescence properties.
Project description:A custom high density oligo-microarray (8 x 15K) was designed and printed by means of the eArray web tool (Agilent) to analyze the transcriptome of the three intestinal sections of Euroipan sea bass (Dicentrarchus labrax). Naïve stock juveniles sea bass, maintained under intensive rearing conditions in the indoor experimental facilities of IATS, were sampled after overnight fasting for anterior, middle and posterior sections of intestine. The array comprised 60-oligomer sequences for 14,147 different sea bass annotated sequences. Total RNA (150ng) from individual fish were labelled with cyanine 3-CTP and 1,000ng of each labelled cRNA were hybridized to microarray slides. Analysis of the scanned data, including principal component analysis and unpaired t-test with Benjamini-Hochberg multiple testing correction, was carried out with GeneSpring GX software (Agilent). Pathway analysis of differentially expressed sequences was performed using the Ingenuity Pathway Analysis (IPA) software.
Project description:Lepeophtheirus salmonis (sea lice) and bacterial co-infection threatens wild and farmed Atlantic salmon performance and welfare. The present microarray-based study examined the dorsal skin transcriptome response to formalin-killed Aeromonas salmonicida bacterin (ASAL) in pre-adult sea lice-infected and non-infected Atlantic salmon to fill the existing knowledge gap and aid in developing anti-co-infection strategies. To this aim, sea lice-infected and non-infected salmon were intraperitoneally injected with either phosphate-buffered saline (PBS) or ASAL (i.e., 4 injection/infection groups: PBS/no lice, PBS/lice, ASAL/no lice, and ASAL/lice). The analysis of the dorsal skin transcriptome data [Significance Analysis of Microarrays (5% FDR)] identified 345 up-regulated and 2,189 down-regulated DEPs in the comparison PBS/lice vs. PBS/no lice, and 82 up-regulated and 3 down-regulated DEPs in the comparison ASAL/lice vs. ASAL/no lice. The comparison ASAL/lice vs. PBS/lice identified 272 up-regulated and 11 down-regulated DEPs, whereas ASAL/no lice vs. PBS/no lice revealed 27 up-regulated DEPs. The skin transcriptome differences between the co-stimulated salmon (i.e., ASAL/lice) and PBS/no lice salmon accounted for 1,878 up-regulated and 3,120 down-regulated DEPs.