Bioluminescent reporter assays are an excellent choice for analyzing gene regulation because they provide higher sensitivity, wider dynamic range and better signal-to-background ratios compared to colorimetric or fluorescent assays. In a typical genetic reporter assay, cells are transfected with a vector that contains the sequence of interest cloned upstream of a reporter gene, and the reporter activity is used to determine how the target sequence influences gene expression under experimental conditions. A second control reporter encoded on the same or a different plasmid is an essential internal control. The secondary reporter is used to normalize the data and compensate for variability caused by differences in cell number, lysis efficiency, cell viability, transfection efficiency, temperature, and measurement time.
For genetic reporter assays, using a secondary control vector with a weak promoter like PGK or TK to ensures that the control does not interfere with activation of your primary reporter vector. Transfection of high amounts of the control plasmid or putting the control reporter under control of a strong promoter like CMV or SV40 often leads to transcriptional squelching or other interference with the experimental promoter (i.e., trans effects). Reporter assays can also be used to quantitatively evaluate microRNA activity by inserting miRNA target sites downstream or 3´ of the reporter gene. For example, the pmirGLO Dual-Luciferase miRNA Target Expression Vector is based on dual-luciferase technology, with firefly luciferase as the primary reporter to monitor mRNA regulation and Renilla luciferase as a control reporter for normalization.
Here in Technical Services we often talk with researchers who are just starting their project and looking for advice on designing their genetic reporter vector. They have questions like:
How much of the upstream promoter region should be included in the vector?
How many copies of a response element will be needed to provide a good response?
Does the location of the element or surrounding sequence alter gene regulation?
These assays are relatively easy to understand in principle. Use a primary and secondary reporter vector transiently transfected into your favorite mammalian cell line. The primary reporter is commonly used as a marker for a gene, promoter, or response element of interest. The secondary reporter drives a steady level of expression of a different marker. We can use that second marker to normalize the changes in expression of the primary under the assumption that the secondary marker is unaffected by what is being experimentally manipulated.
While there are many advantages to dual-reporter assays, they require careful planning to avoid common pitfalls. Here’s what you can do to avoid repeating some of the common mistakes we see with new users:
We rely on insulin supplied by our pancreas at the right dose and at the right time to control our blood glucose levels and energy storage. Insulin works by regulating the energy usage of various cell types in the body. When this process goes awry, it can cause diabetes.
There are two types of diabetes, defined by how insulin is
dysregulated. In Type 1 Diabetes (T1D), the pancreas produces too little
insulin. Patients need to give themselves insulin in order to respond to
glucose in the diet. In Type 2 Diabetes (T2D), patients do not respond well to
the insulin produced in their body. Therefore, they need to give themselves
more to avoid hyperglycemia (high blood glucose).
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.
This summer, I had the opportunity to go to the Marine
Biological Laboratory (MBL) in Woods Hole, Massachusetts. MBL was founded in
1888 as an institution that focuses on research and education. Woods Hole is
located on Cape Cod and has rich biodiversity that is the focus of the resident
researchers and the many others that travel there each summer. It was here that
new model organisms were discovered, allowing significant advancement in
various fields. For example, squid have large axons that allowed researchers to
expand our knowledge of neurons.
Over 500 scientists from over 300 institutions in over 30
countries come to MBL each year as trainees1. There are 19 advanced
research training courses for pre-and post-doctoral scientists in development, reproduction,
cell physiology, microbiology, infectious disease, neuroscience, and microscopy.
Faculty that teach the courses are leaders in their respective fields. In
addition, MBL has a neuro-physiology fellowship program through the Grass
Foundation that allows early-stage researchers to come to MBL for 14 weeks to
Over the past decade, microbiome research has provided key
insights into the relationship between our gut and our health. There are
trillions of organisms in our gut, comprising the microbiome that complements
our human biology, distinct from our genome. These gut microbes affect us in
many ways, from affecting our mental
health to our ability to fight
At the University of Wisconsin-Madison, Federico Rey and his research group are trying to understand how our diet might help or harm the important microbial communities in our gut. “If we can understand how microbes interact with diet, we can personalize nutrition to match diet with the composition of the gut microbiome and promote health,” Rey says.
In recent years, scientists have been hot on the trail of transcription factor FOXO3, tracing its involvement in various tumor-centric activities comprising the many trademarks of cancer, from drug resistance to metastasis to tumor angiogenesis.
FOXO3 is a member of the O sub-class of the forkhead box family of transcription factors. The forkhead box (FOX) family is characterized by a fork head DNA-binding domain (DBD), comprised of around 100 amino acids. They have also proven themselves to be a family of many hats, functioning in diverse roles ranging from metabolism, immunology, cell-cycle control, development, as well as cancer (1). The forkhead box O (FOXO) sub-class alone has demonstrated involvement in a variety of cellular outcomes, from drug resistance and longevity to apoptosis induction.
Due to its pro-apoptotic and anti-proliferative proclivity, FOXO3 has been previously identified as a tumor suppressor gene. However, more and more studies have begun to flip the narrative on FOXO3, portraying it more as a devoted henchman, due to its roles in drug and radiotherapy resistance, cell-cycle arrest and long-term maintenance of leukemia-initiating stem cells in a variety of cancer types, including breast cancer, pancreatic cancer, glioblastoma, and both acute and chronic myeloid leukemia.
As the number of children diagnosed with autism spectrum disorder (ASD) continues to rise, the search for a cause continues. Scientists have been studying genetically modified oxytocin receptors, which have shown promise as a target for studying ASD-related behaviors. One of the obstacles to designing robust scientific experiments for investigating potential ASD causes or treatments is the lack of a truly appropriate model organism for social behaviors in humans (1). Sure, there are the traditional lab rats and lab mice that demonstrate a certain level of social behaviors. However, there has been a loss of natural social behaviors in common lab mice strains because of the reduction in genetic complexity from inbreeding and adaptation to captivity (2). These animals cannot fully represent the depth of human social behaviors, including the ability of humans to form lasting social bonds (1).
Traditionally, scientists have relied on flat,
two-dimensional cell cultures grown on substrates such as tissue culture
polystyrene (TCPS) to study cellular physiology. These models are simple and
cost-effective to culture and process. Within the last decade, however, three-dimensional
(3D) cell cultures have become increasingly popular because they are more
physiologically relevant and better represent in vivo conditions.
You have identified and cloned your protein of interest, but you want to explore its function. A protein fusion tag might help with your investigation. However, choosing a tag for your protein depends on what experiments you are planning. Do you want to purify the protein? Would you like to identify interacting proteins by performing pull-down assays? Are you interested in examining the endogenous biology of the protein? Here we cover the advantages and disadvantages of some protein tags to help you select the one that best suits your needs.
The most commonly used protein tags fall under the category of affinity tags. This means that the tag binds to another molecule or metal ion, making it easy to purify or pull down your protein of interest. In all cases, the tag will be fused to your protein of interest at either the amino (N) or carboxy (C) terminus by cloning into an expression vector. This protein fusion can then be expressed in cells or cell-free systems, depending on the promoter the vector contains. Continue reading “Choosing a Tag for Your Protein”