Shining Stars: Cool NanoLuc® Plasmid Constructs Available Through the Addgene Repository

Researchers having been sharing plasmids ever since there were plasmids to share. Back when I was in the lab, if you read a paper and saw an interesting construct you wished to use, you could either make it yourself or you could “clone by phone”.  One of my professors was excellent at phone cloning with labs around the world and had specific strategies and tactics for getting the plasmids he wanted. Addgene makes this so much easier to share your constructs from lab to lab. Promega supports the Addgene mission statement: Accelerate research and discovery by improving access to useful research materials and information.  Many of our technology platforms like HaloTag® Fusion Protein, codon-optimized Firefly luciferase genes (e.g., luc2), and NanoLuc® Luciferase are present in the repository. We encourage people to go to Addgene to get new innovative tools. Afterall, isn’t science better when we share?

I’d like to focus on some tools in the Addgene collection based on NanoLuc® Luciferase (NLuc).  The creation of NanoLuc® Luciferase and the optimal substrate furimazine is a good story (1).  From a deep sea shrimp to a compact powerhouse of bioluminescence, NLuc is 100-fold brighter than our more common luciferases like firefly (FLuc) and Renilla (RLuc) luciferase.  This is important not so much for how bright you can make a reaction but for how sensitive you can make a reaction.  NLuc requires 100-fold less protein to produce the same amount of light from a Fluc or RLuc reaction.  NLuc lets you work at physiological concentrations.  NLuc is bright enough to detect endogenous tagged genes generated through the CRISPR/Cas9 knock-in.  NLuc is very inviting for endogenous tagging as it is only 19kDa.  An example is the CRISPaint-NLuc construct (Plasmid #67178) for use in the system outlined in Schmid-Burgk, J.L. et al (2).

Two applications of NanoLuc® Technology have caught my attention through coupling the luciferase with fluorescent proteins to make better imaging reporters and biosensors. Continue reading

Luciferase Immunoprecipitation System Assay (LIPS): Expression of Luciferase Antigen using TNT Transcription/Translation Kit

NanoLuc dual reporters

Illustration showing NanoLuc and firefly luciferase reporters.

The luciferase immunoprecipitation system (LIPS) assay is a liquid phase immunoassay allowing high-throughput serological screening of antigen-specific antibodies. The immunoassay involves quantitating serum antibodies by measuring luminescence emitted by the reporter enzyme Renilla luciferase (Rluc) fused to an antigen of interest. The Rluc-antigen fusion protein is recognized by antigen-specific antibodies, and antigen-antibody complexes are captured by protein A/G beads that recognize the Fc region of the IgG antibody (1).

In a recent publication (2), this assay was used to assess the presence of autoantibodies against ATP4A and ATP4B subunits of parietal cells H+, K+-ATPase in patients with atrophic body gastritis and in controls. Continue reading

Surfing the Light Waves: Shrimp, Coral, Turtles and Other Fluorescent Organisms

A branching torch coral, Euphyllia glabrescens.

Have you ever walked on a beach and noticed that the waves seem to glow as they roll onto shore? Perhaps you have seen fish or jellyfish that glow in the dark, or maybe you’ve chased fireflies in your backyard or on a camping trip. These are all forms of luminescence (the production of light without adding heat), but the manner that these organisms produce their light can be quite different. Continue reading

Making BRET the Bright Choice for In vivo Imaging: Use of NanoLuc® Luciferase with Fluorescent Protein Acceptors

13305818-cr-da-nanoluc-application_ligundLive animal in vivo imaging is a common and useful tool for research, but current tools could be better. Two recent papers discuss adaptations of BRET technology combining the brightness of fluorescence with the low background of a bioluminescence reaction to create enhanced in vivo imaging capabilities.

The key is to image photons at wavelengths above 600nm, as lower wavelengths are absorbed by heme-containing proteins (Chu, J., et al., 2016 ). Fluorescent protein use in vivo is limited because the proteins must be excited by an external light source, which generates autofluorescence and has limited penetration due to absorption by tissues. Bioluminescence imaging continues to be a solution, especially firefly luciferase (612nm emission at 37°C), but its use typically requires long image acquisition times. Other luciferases, like NanoLuc, Renilla, and Gaussia, etc. either do not produce enough light or the wavelengths are readily absorbed by tissues, limiting their use to near- surface imaging.

The two papers discussed here illustrate how researchers have combined NanoLuc® luciferase with a fluorescent protein to harness bioluminescent resonance energy transfer (BRET) for brighter in vivo imaging reporters. Continue reading

Don’t Let These Three Common Issues Hurt Your Luminescent Assay Results

4621CAThere is a lot riding on your luminescent assay results. Each plate represents precious time, effort and resources. Did you know that there are three things about your detection instrument that can impact how much useful information you get from each plate?  Instruments with poor sensitivity may cause you to miss low-level samples that could be the “hit” you are looking for.  Instruments with a narrow detection range limit the accuracy or reproducibility you needed to repeat your work.  Finally, instruments that let the signal from bright wells spill into adjacent wells allow crosstalk to occur and skew experimental results, costing you time and leading to failed or repeated experiments. Continue reading

Making the Switch from FRET to BRET: Applications of NanoLuc® Luciferase with Fluorescent Protein Acceptors for Sensing Cellular Events

A Bioluminescent Alternative

Fluorescence resonance energy transfer (FRET) probes or sensors are commonly used to measure cellular events. The probes typically have a matched pair of fluorescent proteins joined by a ligand-binding or responsive protein domain. Changes in the responsive domain are reflected in conformational changes that either bring the two fluorescent proteins together or drive them apart. The sensors are measured by hitting the most blue-shifted fluorescent protein with its excitation wavelength (donor). The resulting emission is transferred to the most red-shifted fluorescent protein in the pair, and the result is ultimately emission from the red-shifted protein (acceptor).

As pointed out by Aper, S.J.A. et al. below, FRET sensors face challenges of photobleaching, autofluorescence, and, in the case of exciting cyan-excitable donors, phototoxicity. Another challenge to using FRET sensors comes when employing optogenetic regulators to initiate the event you wish to monitor. Optogenetic regulators respond to specific wavelengths and initiate signaling. The challenge comes when the FRET donor excitation overlaps with the optogenetic initiation wavelengths. Researchers have sought to alleviate many of these challenges by exchanging the fluorescent donor for a bioluminescent donor, making bioluminescence resonance energy transfer (BRET) probes. In the three papers described below, the authors chose NanoLuc® Luciferase as the BRET donor due to its extremely bright signal. Continue reading

The Role of the NanoLuc® Reporter in Investigating Ligand-Receptor Interactions

Luminescent reporter assays are powerful research tools for a variety of applications. Last March we presented a webinar on this topic, Understanding Luminescent Reporter Assay Design, which proved to enlighten many who registered. The webinar addressed the importance of careful experimental design when using a luminescent reporter such as Promega’s Firefly or NanoLuc® Luciferase.

Reporters provide a highly sensitive, quantifiable metric for cellular events such as gene expression, protein function and signal transduction. Luminescent reporters have become even more valuable for live, real-time measurement of various processes in living cells. This is backed by the fact that a growing number of scientific publications reference the use of the NanoLuc® Luciferase reporter and demonstrate its effectiveness as a reporter assay. Continue reading

Bioassay for Cannabinoid Receptor Agonists Designed with NanoBiT™ Techology

Cannabinoids. What are they? Sometimes, Wikipedia can give a nice definition:

Tetrahydrocannabinol (THC), a partial agonist of the CB1 and CB2 cannabinoid receptors. Wikipedia Commons

Tetrahydrocannabinol (THC), a partial agonist of the CB1 and CB2 cannabinoid receptors. Wikipedia Commons

A cannabinoid is one of a class of diverse chemical compounds that acts on cannabinoid receptors in cells that alter neurotransmitter release in the brain. Ligands for these receptor proteins include the endocannabinoids (produced naturally in the body by animals), the phytocannabinoids (found in Cannabis and some other plants), and synthetic cannabinoids (manufactured artificially).

Synthetic cannabinoids (SCs) were originally created for the scientific investigation of two cannabinoid receptors, CB1 and CB2, but have made their way to the streets as “safe” and “legal” alternatives to marijuana.

The problem is that these SCs engage the cannabinoid receptors more completely and with higher affinity than anything derived from marijuana. As a result, SCs can produce serious side effects that often require medical attention. In fact, you are 30 times more likely to seek emergency medical attention following the use of an SC than with natural cannabinoid sources like marijuana. Continue reading

Improving the Success of Your Transfection

12150558-plasmid_with_cell_membrane3Not every lab has a tried and true transfection protocol that can be used by all lab members. Few researchers will use the same cell type and same construct to generate data. Many times, a scientist may need to transfect different constructs or even different molecules (e.g., short-interfering RNA [siRNA]) into the same cell line, or test a single construct in different cultured cell lines. One construct could be easily transfected into several different cell lines or a transfection protocol may work for several different constructs. However, some cells like primary cells can be difficult to transfect and some nucleic acids will need to be optimized for successful transfection. Here are some tips that may help you improve your transfection success.

Transfect healthy, actively dividing cells at a consistent cell density. Cells should be at a low passage number and 50–80% confluent when transfected. Using the same cell density reduces variability for replicates. Keep cells Mycoplasma-free to ensure optimal growth.

Transfect using high-quality DNA. Transfection-quality DNA is free from protein, RNA and chemical contamination with an A260/A280 ratio of 1.7–1.9. Prepare purified DNA in sterile water or TE buffer at a final concentration of 0.2–1mg/ml. Continue reading

Shining Light on a Superbug

Antibiotic-resistant bacteria and their potential to cause epidemics with no viable treatment options have been in the news a lot. These “superbugs,” which have acquired genes giving them resistance to common and so-called “last resort” antibiotics, are a huge concern as effective treatment options dwindle. Less attention has been given to an infection that is not just impervious to antibiotics, but is actually enabled by them.

Clostridium diffic33553646_lile Infection (CDI) is one of the most common healthcare-associated infections and a significant global healthcare problem. Clostridium difficile (C. diff), a Gram-positive anaerobic bacterium, is the source of the infection. C. diff spores are very resilient to environmental stressors, such as pH, temperature and even antibiotics, and can be found pretty much everywhere around us, including on most of the food we eat. Ingesting the spores does not usually lead to infection inside the body without also being exposed to antibiotics.

Individuals taking antibiotics are 7-10 times more likely to acquire a CDI. Antibiotics disrupt the normal flora of the intestine, allowing C. diff to compete for resources and flourish. Once exposed to the anaerobic conditions of the human gut, these spores germinate into active cells that embed into the tissue lining the colon. The bacteria are then able to produce the toxins that can cause disease and result in severe damage, or even death. Continue reading