Post 3 Apr 27, 2011

"I am that merry wanderer of the night" 

- A Midsummer Nights Dream

Luciferase is the enzyme which is used by fire flies to glow.  It uses the substrate luciferin for this oxidative process.  The reaction is as seen below:

University of Wisconsin, Dana Nystrom, http://people.uwec.edu/nystrodl/chem.htm

So what are the advantages to using Luciferase versus traditional practices?

• highly sensitive only 1 x10^5 luciferase molecules needed for detection.
• little background noise
•  non toxic
• *cheap and easy to use 

This picture shows the overall structure with the Luciferyl AMP in the center.

A close up of the main molecule

 

 The opening shows how the substrate enters the enzyme. This is the opening to the active site.

What are things being done with Luciferase? 

Bifunctional Antibody-Renilla Luciferase Fusion Protein for In Vivo Optical Detection of Tumors


Renilla Luciferase (R-Luc) produces light upon mechanical stimulation. In this study R-Luc is fused to an antibody for cancer cells. The antibody used is Carinoembryonic antigen (CEA) was because it is highly expressed in colon cancer and is a surface marker. Fusion protein was tested for for binding to cancer cells through flow cytometry. The fusion protein was tested for expression and stability using bacterial periplasmic secretions and stability was improved by mutagenesis.

The protein was injected into a mouse under anesthesia, through the tail and PET and Optical imaging was used to capture the light emitted and the target tumor. The result was is as shown in the below picture:

This experiment allows for R-Luc to be fused to other known disease antibodies. With this kind of information, specialized treatment can be developed, and more about the disease can be known.  The advantages of this method are decreased background signaling resulting in higher sensitivity and more specificity. Extracellular targets as well as intracellular targets are in clinical studies.

Phase-Specific Circadian Clock Regulatory Elements in Arabidopsis

In this experiment, arabidopsis  was transformed with Luciferase to study the Circadian biological clock. That is in plants, stoma opening and leaf movements are dependent on the biological clocks. The transformed arabidopsis were exposed to environmental conditions such as temperature and light/no light. Luciferase activity was then measured to help determine the activity of the plant. The experiment also included variations of the promoter region to determine which ones were important for circadian rhythms. They also were able to figure out which regions dealt with morning rhythms versus night rhythms. These rhythms were found to be most important on the transcriptional level. 

http://www.clt.astate.edu/mhuss/lecture_notes_set_no__5.htm

 With the ability to transform plants to glow like this, will Christmas Trees be next? 

What other Organisms use Luciferases?

Figure 1. Pinecone fish utilize luminous bacteria, colonized in the ventral cavity, to illuminate the surroundings as well as for intra-species communication. 

Figure 2. The deep sea Angler fish carries luminous bacteria in a light emitting rod, which attracts prey to the front of its mouth.

Figure 3. Luminous bacteria reside in symbiosis on a pair of light organs in the mantle body of the squid. Utilization of the illumination function is believed to frighten nearby predators, allowing the squid to escape.

(http://www.photobiology.info/Lin.html)

Figure 4. ET, our extraterrestrial friend has been rumored to also have luciferase at the tips of his
fingers. :D




Bifunctional Antibody-Renilla Luciferase Fusion Protein for In Vivo Optical Detection of Tumors
by Katy M.Venisnik, Tove Olafsen, Andreas M.Loening, Meera Iyer, Sanjiv S.Gambhir and Anna M.Wu1,
read online

Phase-Specific Circadian Clock Regulatory Elements in Arabidopsis

Todd P. Michael and C. Robertson McClung

read online

ASSN 2-journals

"I am that merry wanderer of the night" 

- A Midsummer Nights Dream

Luciferase is the enzyme which is used by fire flies to glow.  It uses the substrate luciferin for this oxidative process.  The reaction is as seen below:

University of Wisconsin, Dana Nystrom, http://people.uwec.edu/nystrodl/chem.htm

So what are the advantages to using Luciferase versus traditional practices?

• highly sensitive only 1 x10^5 luciferase molecules needed for detection.
• little background noise
•  non toxic
• *cheap and easy to use

Here are three journal summaries which relate to research using luciferase:

Bifunctional Antibody-Renilla Luciferase Fusion Protein for In Vivo Optical Detection of Tumors

by Katy M.Venisnik, Tove Olafsen, Andreas M.Loening, Meera Iyer, Sanjiv S.Gambhir and Anna M.Wu1,
read online


Renilla Luciferase (R-Luc) produces light upon mechanical stimulation. In this study R-Luc is fused to an antibody for cancer cells. The antibody used is Carinoembryonic antigen (CEA) was because it is highly expressed in colon cancer and is a surface marker. Fusion protein was tested for for binding to cancer cells through flow cytometry. The fusion protein was tested for expression and stability using bacterial periplasmic secretions and stability was improved by mutagenesis.

The protein was injected into a mouse under anesthesia, through the tail and PET and Optical imaging was used to capture the light emitted and the target tumor. The result was is as shown in the below picture:

This experiment allows for R-Luc to be fused to other known disease antibodies. With this kind of information, specialized treatment can be developed, and more about the disease can be known.  The advantages of this method are decreased background signaling resulting in higher sensitivity and more specificity. Extracellular targets as well as intracellular targets are in clinical studies.

Development of Functional Genomic Tools in Trematodes: RNA Interference and Luciferase Reporter Gene Activity in Fasciola hepatica

Gabriel Rinaldi^1,2, Maria E. Morales¹, Martín Cancela², Estela Castillo³, Paul J. Brindley^1¤, José F. Tort^2*

read online 

 RNAi is a potential way to treat many disease causing parasites because it targets  mRNA. This interferes with protein production. In this article, researchers used reverse genetic tools.  The parasitic F.hepatica was electroporated with mRNA of Luciferase. Once the activity of Luciferase was confirmed the researchers electroporated the parasites again with dsRNA. If there was a RNAi pathway the luciferase activity would be silenced. With this method, gene function investigations can be enhanced in Fasciola parasites. Specific targets can also be investigated. This also shows a new path for genetic manipulation using RNAi and less known trematodes can be studied further.

Phase-Specific Circadian Clock Regulatory Elements in Arabidopsis

Todd P. Michael and C. Robertson McClung

read online

In this experiment, arabidopsis  was transformed with Luciferase to study the Circadian biological clock. That is in plants, stoma opening and leaf movements are dependent on the biological clocks. The transformed arabidopsis were exposed to environmental conditions such as temperature and light/no light. Luciferase activity was then measured to help determine the activity of the plant. The experiment also included variations of the promoter region to determine which ones were important for circadian rhythms. They also were able to figure out which regions dealt with morning rhythms versus night rhythms. These rhythms were found to be most important on the transcriptional level.

The figure below is not from the journal but it was one of the earlier transformed tobacco plants with Luciferase:

http://www.clt.astate.edu/mhuss/lecture_notes_set_no__5.htm

 

 

 

 

 With the ability to transform plants to glow like this, will Christmas Trees be next? 

Assn1-Luciferase...

 "Hope will come to me like a firefly.

In my hour of ebony fog, 

Burning with luminosity,

From no where else but God"

- Gwendolyn Taylor Soper

The firefly emits a light in the dark of night in order to attract prey and mates. Who can forget those warm summer nights watching the sunset and waiting for the fireflies to come out. So what is it that makes the fireflies glow? 

The firefly uses  the enzyme Luciferase which uses  Magnesium or Chloride ions, ATP, and oxygen to create light.