Bioluminescent Reporters Bioluminescent Reporters Gene Expression Andrew Costello
Bioluminescent Reporters
Bioluminescent Reporters Gene Expression Andrew Costello
Green Fluorescent Protein
Green Fluorescent Protein �First isolated in Aequorea victoria �Used to help identify certain proteins �Studies resulting from GFP research: Developmental regulation of Candida albicans promoters Protein localization
Green Fluorescent Protein �In Candida albicans, promoters WH 11, EF 1 a 2, GAL 1, and OP 4 are tagged by GFP �In order to integrate the GFP into genome, plasmid is formed �For RLUC to be expressed, the sequence must be upstream of the promoter
Construction of Plasmid
Purpose of Experiment �Helps determine which promoter is expressed under certain conditions � By knowing which promoter is expressed under certain conditions, can target the promoter and stop its transcription
Protein Localization �GFP helps to localize the sequence of proteins in pathogens �Previously, genetic analyses depended on reverse genetics to identify the genome
Protein Localization
Protein Localization �y. EGFP 3 is prepared and integrated into the genome of bacteria �Overall, the proteins fluoresced �Other reporter systems β-galactosidase Luciferase
Purpose of Experiment �y. EGFP 3 helps identify proteins in C. albicans and S. cerevisiae Both are pathogenic to humans and GFP helps detect these pathogens �y. EGFP 3 is more ideal for gene expression Monitored at each cell Maintains cell viability
Bioluminescent Reporters Post-Transcriptional Modification Patrick Suess
Post-Transcriptional Modification �Primary Transcript RNA Mature RNA
Codon Optimization �Native luciferase genes not optimal in mammalian cells �Alter codons increase in luciferase levels
Degradation Signal Addition �Assays measure total accumulation of reporter �Affected by m. RNA stability �Degradation signals PEST and CL 1
Example of Research �RNA interference Study how ds. RNA suppresses expression if target m. RNA Luciferase reporters used to quantitatively analyze micro. RNA activity by insertion of target sites downstream of firefly luciferase gene
Protein-Protein Interactions �Chris Garza
Renilla reniformis – Sea Pansy
Bioluminescence Resonance Energy Transfer (BRET) �Naturally occurring in Renilla reniformis A variation of Förster resonance energy transfer (FRET) ▪ FRET – A colored molecule (donor) excites an acceptor ▪ Acceptor gives off a different color ▪ BRET – A luminescent donor and a fluorescent acceptor Renilla Luciferase (Rluc) = donor Green Fluorescent Protein = acceptor ▪ Emits a green light ▪ Used in science to study Protein-Protein Interactions
Protein-Protein Interaction Assays � Proteins are fused to the luciferase donor and a GFP (or YFP) acceptor Donor excites the acceptor if the proteins interact ▪ Light given off showing protein-protein interactions � Used for receptor and signal transduction pathways research
Examples of Research �NMDA receptor and PSD-95 scaffolding protein fused to Rluc and GFP (Gottschalk 2009) Potential in finding a treatment for ischemic brain disease �Transgenic mice producing fused proteins (Audet 2010) Beta(2)-adrenergic receptor fused with Rluc Beta arrestin-2 fused with GFP
References � Gottschalk M, Bach A, Hansen JL, Krogsgaard-Larsen P, Kristensen AS, Tromgaard, K. 2009. Detecting protein-protein interactions in living cells: development of a bioluminescence resonance energy transfer assay to evaluate the PSD-95/NMDA receptor interaction. Neurochem Research. 34(10): 1729 -1737. � Audet M, Lagacé M, Silversides DW, Bouvier M. 2010. Protein-protein interactions monitored in cells from transgenic mice using bioluminescence resonance energy transfer. Faseb Journal. 24(8): 2829 -2838. � Loening AM, Fenn TD, Gambhir SS. 2007. Crystal Structures of the Luciferase and Green Fluorescent Protein from Renilla reniformis. Journal of Molecular Biology. 374(4): 1017 -1028. � BMG Labtech. BRET - a new method for assaying protein-protein interactions in living cells. Pharmaceutical International. � Promega- protocols and applications guide. 2009. Bioluminescent Reporters. http: //www. promega. com/paguide/chap 8. htm � Andreu N, Zelmer A, Fletcher T, Elkington PT, Ward TH, Ripoll J, Parish T, Bancroft GJ, Schaible U, Robertson BD, Wiles S. Optimisation of bioluminescent reporters for use with mycobacteria. PLo. S One. 2010 May 24; 5(5): e 10777.
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