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 WH11, EF1a2, GAL1, and OP4 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 yEGFP3 is prepared and integrated into the genome of bacteria Overall, the proteins fluoresced Other reporter systems β-galactosidase Luciferase

Purpose of Experiment yEGFP3 helps identify proteins in C. albicans and S. cerevisiae Both are pathogenic to humans and GFP helps detect these pathogens yEGFP3 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 Methylated guanine cap to 5 prime end, poly-A tail to other end, introns spliecd out, exons spliced together. Ready for translation

Codon Optimization Native luciferase genes not optimal in mammalian cells Alter codons increase in luciferase levels Increase in levels make it easier to detect in assays

Degradation Signal Addition Assays measure total accumulation of reporter Affected by mRNA stability Degradation signals PEST and CL1 Increase stability causes accumulation of reporter in higher levels, but concentration changes very slowly relative to changes in transcription rate. Decreased Stability accumulates in lower levels, but increases the rate of response with changes in transcription PEST and CL1 are degradation signals used in firefly lucifersase

Example of Research RNA interference Study how dsRNA suppresses expression if target mRNA Luciferase reporters used to quantitatively analyze microRNA activity by insertion of target sites downstream of firefly luciferase gene

Protein-Protein Interactions Chris Garza

Renilla reniformis – Sea Pansy Renilla reniformis exists as a collection of marine polyps that work together to form what is commonly known as a sea pansy. One polyp forms the peduncle which is this anchoring foot. The other polyps the petal-like structure. The polyps with tentacles do the feeding while other polyps work to inflate or deflate the structure. The sea pansy’s greatest contribution to science is its naturally occurring ability to bioluminesce through a process called BRET.

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 BRET stands for Bioluminescence Resonance Energy Transfer. BRET is a variation of FRET or Forster Resonance Energy Transfer, named after the German scientist Theodor Forster. In FRET, a molecule of a certain color (donor) excites another molecule (acceptor) so that it will give off another color. BRET differs from FRET in that the donor is luminescent and the acceptor is fluorescent. In Renilla reniformis, the donor is a luciferase and the acceptor is a Green Fluorescent Protein. The luciferase gives off a blue color and excites the GFP to give off a green light. This has been taken advantage of 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 Scientists have fused proteins to luciferase and some sort of GFP variant to study protein-protein interactions. If the proteins come within 1-10 nm regions of each other the donor excites the acceptor and the wavelength of light can be detected. This assay method can be used to better understand protein-protein interactions in receptor research and signal transduction pathways among other things.

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 An experiment was done to detect interactions between the NMDA receptor and the PSD-95 scaffolding protein. The researchers fused Rluc and GFP to these proteins and successfully measured the light given off. Understanding the interactions between these proteins has potential in finding a treatment for ischemic heart disease. In another experiment, researchers designed transgenic mice that would express fused proteins. They fused a beta(2)-adrenergic and Renilla luciferase together as well as a beta arrestin-2 fused to a green fluorescent protein. The researchers achieved their goal of producing the transgenic mice that could be used to study protein-protein interactions.

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/chap8.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. PLoS One. 2010 May 24;5(5):e10777.