1. Bioluminescent Reporters

2. Bioluminescent Reporters
Gene Expression
Andrew Costello

3. Green Fluorescent Protein

4. 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

5. 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

6. Construction of Plasmid

7. 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

8. Protein Localization
GFP helps to localize the sequence of proteins in pathogens
Previously, genetic analyses depended on reverse genetics to identify the genome

9. Protein Localization

10. Protein Localization
yEGFP3 is prepared and integrated into the genome of bacteria
Overall, the proteins fluoresced
Other reporter systems
β-galactosidase
Luciferase

11. 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

12. Bioluminescent Reporters
Post-Transcriptional Modification
Patrick Suess

13. 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

14. 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

15. 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

16. 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

17. Protein-Protein Interactions
Chris Garza

18. 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.

19. 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.

20. 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.

21. 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.

22. References
Gottschalk M, Bach A, Hansen JL, Krogsgaard-Larsen P, Kristensen AS, Tromgaard, K 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):
Audet M, Lagacé M, Silversides DW, Bouvier M Protein-protein interactions monitored in cells from transgenic mice using bioluminescence resonance energy transfer. Faseb Journal. 24(8):
Loening AM, Fenn TD, Gambhir SS Crystal Structures of the Luciferase and Green Fluorescent Protein from Renilla reniformis. Journal of Molecular Biology. 374(4):
BMG Labtech. BRET - a new method for assaying protein-protein interactions in living cells. Pharmaceutical International.
Promega- protocols and applications guide Bioluminescent Reporters.
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 May 24;5(5):e10777.