Bioluminescent Reporters Bioluminescent Reporters Gene Expression Andrew Costello

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Bioluminescent Reporters

Bioluminescent Reporters

Bioluminescent Reporters Gene Expression Andrew Costello

Bioluminescent Reporters Gene Expression Andrew Costello

Green Fluorescent Protein

Green Fluorescent Protein

Green Fluorescent Protein �First isolated in Aequorea victoria �Used to help identify certain proteins

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

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

Construction of Plasmid

Purpose of Experiment �Helps determine which promoter is expressed under certain conditions � By

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

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

Protein Localization �y. EGFP 3 is prepared and integrated into the genome of bacteria

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.

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

Bioluminescent Reporters Post-Transcriptional Modification Patrick Suess

Post-Transcriptional Modification �Primary Transcript RNA Mature RNA

Post-Transcriptional Modification �Primary Transcript RNA Mature RNA

Codon Optimization �Native luciferase genes not optimal in mammalian cells �Alter codons increase in

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 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.

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

Protein-Protein Interactions �Chris Garza

Renilla reniformis – Sea Pansy

Renilla reniformis – Sea Pansy

Bioluminescence Resonance Energy Transfer (BRET) �Naturally occurring in Renilla reniformis A variation of Förster

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

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

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.

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.