Bioluminescent Sharks Set the Sea Aglow

Many deep sea creatures are bioluminescent. However, before documenting the luminescence of the kitefin shark, Dalatias licha, there has never been a nearly six-foot long luminous vertebrate creature. In a recent study, Mallefet and colleagues examined three species of sharks: Dalatias licha, Etmopterous lucifer, and Emopterus granulosus and documented their luminescence for the first time. These bioluminescent sharks are the largest bioluminescent creatures known.

Researchers studied three species of bioluminescent sharks near the Chatham Islands, New Zealand
Coastline of one of the Chatham Islands, New Zealand
Continue reading “Bioluminescent Sharks Set the Sea Aglow”

Advancing Understanding of Hypoxic Gene Regulation Using Reporter Genes: Celebrating the Work of Dr. Gregg L. Semenza

This post is written by guest blogger, Amy Landreman, PhD, Sr. Product Manager at Promega Corporation.

Oxygen is necessary for animal life. It’s essential for cellular respiration and the production of energy (ATP) we require to survive. Given the need for oxygen, it isn’t surprising that our bodies have evolved ways to sense and adapt to decreased oxygen conditions (hypoxia). We can increase the production of new blood vessels by producing vascular endothelial growth factor (VEGF) or increase red blood cell (RBC) production by increasing the levels of eythropoietin (EPO), the hormone that plays a key role in the production of RBCs. But how does our body sense low oxygen, increase EPO levels, and kick our RBC production into gear? Nobel laureate Gregg L. Semenza has been honored for his contributions to our understanding of this process, and his research demonstrates the value of reporter genes and bioluminescence for studying gene regulation.

Reporter genes and bioluminescence are important tools for studying gene regulation
Continue reading “Advancing Understanding of Hypoxic Gene Regulation Using Reporter Genes: Celebrating the Work of Dr. Gregg L. Semenza”

A Closer Look at C. difficile Biology with Luminescent Tagging

Clostridium difficile is a bacterium that infects around half a million people per year in the United States. The infection causes symptoms ranging from diarrhea to severe colitis, and it’s one of the most common infections contracted while staying in the hospital. As the global incidence of C. diff infection has risen over the past decade, so has the pressure to develop novel therapeutic strategies. This requires a thorough exploration of all aspects of C. difficile biology.

Two recent papers published by researchers at the University of Leiden have shed light on C. difficile physiology using HiBiT protein tagging. The HiBiT system allows detection of proteins in live cells using an 11 amino acid tag. The HiBiT tag binds to the complementary LgBiT polypeptide to reconstitute the luminescent NanoBiT® enzyme. The resulting luminescence is proportional to the amount of HiBiT-tagged protein that is present.

Continue reading “A Closer Look at C. difficile Biology with Luminescent Tagging”

NanoLuc® Luciferase: Brighter Days Ahead for In Vivo Imaging

nanoluc in vivo imaging

The development of NanoLuc® luciferase technology has provided researchers with new and better tools to study endogenous biology: how proteins behave in their native environments within cells and tissues. This small (~19kDa) luciferase enzyme, derived from the deep-sea shrimp Oplophorus gracilirostris, offers several advantages over firefly or Renilla luciferase. For an overview of NanoLuc® luciferase applications, see: NanoLuc® Luciferase Powers More than Reporter Assays.

The small size of NanoLuc® luciferase, as well the lack of a requirement for ATP to generate a bioluminescent signal, make it particularly attractive as a reporter for in vivo bioluminescent imaging, both in cells and live animals. Expression of a small reporter molecule as a fusion protein is less likely to interfere with the biological function of the target protein. NanoLuc® Binary Technology (NanoBiT®) takes this approach a step further by creating a complementation reporter system where one subunit is just 11 amino acids in length. This video explains how the high-affinity version of NanoBiT® complementation (HiBiT) works:

Continue reading “NanoLuc® Luciferase: Brighter Days Ahead for In Vivo Imaging”

NanoLuc: Tiny Tag with a Big Impact

Synthetic biology—genetically engineering an organism to do or make something useful—is the central goal of the iGEM competition each year. After teams conquer the challenge of cloning their gene, the next hurdle is demonstrating that the engineered gene is expressing the desired protein (and possibly quantifying the level of expression), which they may do using a reporter gene.

Reporters can also play a more significant role in iGEM projects when teams design their organism with reporter genes to detect and signal the presence of specific molecules, like environmental toxins or biomarkers. Three of the iGEM teams Promega sponsored this year opted to incorporate some version of NanoLuc® Luciferase into their projects.

NanoLuc® luciferase is a small monomeric enzyme (19.1kDa, 171 amino acids) based on the luciferase from the deep sea shrimp Oplophorus gracilirostris. This engineered enzyme uses a novel substrate, furimazine, to produce high-intensity, glow-type luminescence in an ATP-independent reaction. Unlike other molecules for tagging and detecting proteins, NanoLuc® luciferase is less likely to interfere with enzyme activity and affect protein production due to its small size.

NanoLuc® Luciferase has also been engineered into a structural complementation reporter system, NanoBiT® Luciferase, that contains a Large subunit (LgBiT) and two small subunit options: low affinity SmBiT and high affinity HiBiT. Together, these NanoLuc® technologies provide a bioluminescent toolbox that was used by the iGEM teams to address a diverse set of biological challenges.

Here is an overview of each team’s project and how they incorporated NanoLuc® technology.

Continue reading “NanoLuc: Tiny Tag with a Big Impact”

WiSciFest 2019: A Retrospective

This past weekend was the 9th Annual Wisconsin Science Festival, and we at Promega were excited to join in the celebration of science throughout the state. We participated in the Discovery Expo on Thursday and Friday, where dozens of demonstrations and exhibits were scattered throughout the Wisconsin Institute for Discovery building. Thousands of children on field trips filled the halls, eager to poke and prod at strange and exciting new things.

At our table, we talked about the science of bioluminescence. With 3D-printed firefly luciferase models in hand, we showed the glow of recombinant luciferase to the incoming children and explained to them how scientists could use bioluminescence like a tiny “flashlight” to look inside of cells and watch what’s happening. Our learners received a nice little reward for their attentiveness in the form of glow-in-the-dark firefly stickers.

Thank you to the tablecloth for providing the darkness needed to grab this pic.
Continue reading “WiSciFest 2019: A Retrospective”

Striking Fear into the Heart of Cardiovascular Disease Using Zebrafish and NanoLuc® Luciferase

Representative images of ApoB-LP localization in zebrafish across developmental, genetic, pharmacological and dietary manipulations.
Credit: Figure 5.D of The LipoGlo reporter system for sensitive and specific monitoring of atherogenic lipoproteins by James Thierer, Stephen C. Ekker and Steven A. Farber.
Article licensed under Creative Commons Attribution 4.0 International License.

Cardiovascular diseases, or CVDs, are collectively the most notorious gang of cold-blooded killers threatening human lives today. These unforgiving villains, including the likes of coronary heart disease, cerebrovascular disease and pulmonary embolisms, are jointly responsible for more deaths per year than any other source, securing their seat as the number one cause of human mortality on a global scale.

One of the trademarks of most CVDs is the thickening and stiffening of the arteries, a condition known as atherosclerosis. Atherosclerosis is characterized by the accumulation of cholesterol, fats and other substances, which together form plaques in and on the artery walls. These plaques clog or narrow your arteries until they completely block the flow of blood, and can no longer supply sufficient blood to your tissues and organs. Or the plaques can burst, setting off a disastrous chain reaction that begins with a blood clot, and often ends with a heart attack or stroke.

Given the global prevalence and magnitude of this problem, there is a significant and urgent demand for better ways to treat CVDs. In a recent study published in Nature Communications, researchers at the Carnegie Institution for Science, Johns Hopkins University and Mayo Clinic are taking the fight to CVDs through the study of low-density lipoproteins (LDLs), the particles responsible for shuttling bad cholesterol throughout the bloodstream.

Continue reading “Striking Fear into the Heart of Cardiovascular Disease Using Zebrafish and NanoLuc® Luciferase”

Announcing the 2019 Promega iGEM Grant Winners

It’s FINALLY time to announce the winners of the 2019 Promega iGEM Grant! We received over 150 applications this year, so picking the top 10 was very tough. As always, we’re impressed by the amazing work iGEM teams are doing in the lab and in their communities. The 10 winners listed below will receive $2,000 in free Promega products.

Good luck to all teams competing in iGEM this year, and congratulations to our winners! Don’t forget that Promega has free technical support for all teams competing in iGEM. Our scientists are excited to help out. You can also check out our iGEM Sponsor page, which has tools and resources to help make your project a success. Continue reading “Announcing the 2019 Promega iGEM Grant Winners”

When Proteins Get Together: Shedding (Blue) Light on Cellular LOV

NanoBRETNo protein is an island. Within a cell, protein-protein interactions (PPIs) are involved in highly regulated and specific pathways that control gene expression and cell signaling. The disruption of PPIs can lead to a variety of disease states, including cancer.

Two general approaches are commonly used to study PPIs. Real-time assays measure PPI activity in live cells using fluorescent or luminescent tags. A second approach includes methods that measure a specific PPI “after the fact”; popular examples include a reporter system, such as the classic yeast two-hybrid system.

Continue reading “When Proteins Get Together: Shedding (Blue) Light on Cellular LOV”

Eight Considerations for Getting the Best Data from Your Luminescent Assays

The stage is set. You’ve spent days setting up this experiment. Your bench is spotless. All the materials you need to finally collect data are laid neatly before you. You fetch your cells from the incubator, add your detection reagents, and carefully slide the assay plate into the luminometer. It whirs and buzzes, and data begin to appear on the computer screen. But wait!

Bad data
These data are garbage!

Don’t let this dramatic person be you. Here are 8 tips from us on things to watch out for before you start your next luminescent assay. Make sure you’ll be getting good data before wasting precious sample!

Continue reading “Eight Considerations for Getting the Best Data from Your Luminescent Assays”