Biotechnology From the Mouths of Babes

As a science writer, much of my day entails reviewing and revising marketing materials and technical literature about complex life science research products. I take for granted the understanding that I, my colleagues and our customers have of how these technologies work. This fact really struck me as I read an article about research to improve provider-patient communication in healthcare settings.

The researchers completed an analysis revealing that patient information materials had an average readability at a high school level, while the average patient reads at a fourth-grade level. These findings inspired the researchers to conduct a study in which they enlisted the help of elementary students to revise the content of the patient literature after giving them a short lesson on the material.

The resulting content did not provide more effective ways to communicate indications, pre- and post-op care, risks or procedures—that wasn’t really the point. Instead, the study underscores the important connection between patient literacy and health outcomes. More specifically, a lack of health literacy is correlated with poor outcomes and increased healthcare costs, prompting action from the US Department of Health & Human Services.

While healthcare information can be complex and full of specific medical terminology, I recognized that a lot of the technical and marketing information we create for our products at Promega has similar features. Wouldn’t it be interesting to find out how descriptions of some of our biggest technologies translate through the eyes and mouths of children?

After enlisting some help from my colleagues, I was able to catch a glimpse of how our complex technologies are understood by the little people in our lives. The parents and I explained a technology and then had our child provide a description or drawing of what they understood. Continue reading

Top 5 Most Read Promega Papers in 2017

It’s always nice to know that someone is reading your paper. It’s a sign that your research is interesting, useful and actually has an impact on the scientific community. We were thrilled to learn that papers published by Promega scientists made the top 10 most read papers of 2017 in the journal ACS Chemical Biology. In fact, Promega scientists authored five of the top six most read papers! Let’s take a look at what they are.

#5 CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide

Publication Date (Web): September 11, 2017

This 2017 paper introduces our newest star: HiBiT, a tiny 11aa protein tag. To any scientist studying endogenous protein expression, the HiBiT Tagging System is your dream come true. It combines quantitative and highly sensitive luminescence-based measurement with a tiny-sized tag that can be easily inserted into endogenous protein via CRISPR/Cas9 gene editing with little impact on native protein function. The HiBiT Tagging System has been listed as a 2017 Top 10 Innovation by The Scientist, and it will drastically change how we study endogenous protein expression. 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

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

Two light stories for Friday

For this Friday blog, here’s a sampling of two recent papers highlighting use of the small, bright, NanoLuc luciferase in interesting ways.

Bioluminescence-based hormone:receptor binding studies

A review by Ya-Li Liu and Zhan-Yun Guo, published this week in Amino Acids summarizes recent work of the authors and others using NanoLuc luciferase labeled protein/peptide hormones in receptor binding assays. Typically, studies assessing binding of hormones to receptors have used radioactive tracers. The brightness of NanoLuc luciferase makes bioluminescence an attractive alternative as a sensitive and safer option. Because cell membrane receptors are difficult to purify in quantity, the amounts available for experiments are usually limited. Therefore, tracers used in binding assays need to have a high affinity for the receptor, must not interfere with binding, and must be highly sensitive. Continue reading

For Protein Complementation Assays, Design is Everything

Most, if not all, processes within a cell involve protein-protein interactions, and researchers are always looking for better tools to investigate and monitor these interactions. One such tool is the protein complementation assay (PCA). PCAs use  a reporter, like a luciferase or fluorescent protein, separated into two parts (A and B) that form an active reporter (AB) when brought together. Each part of the split reporter is attached to one of a pair of proteins (X and Y) forming X-A and Y-B. If X and Y interact, A and B are brought together to form the active enzyme (AB), creating a luminescent or fluorescent signal that can be measured. The readout from the PCA assay can help identify conditions or factors that drive the interaction together or apart.

A key consideration when splitting a reporter is to find a site that will allow the two parts to reform into an active enzyme, but not be so strongly attracted to each other that they self-associate and cause a signal, even in the absence of interaction between the primary proteins X and Y. This blog will briefly describe how NanoLuc® Luciferase was separated into large and small fragments (LgBiT and SmBiT) that were individually optimized to create the NanoBiT® Assay and show how the design assists in monitoring protein-protein interactions.

Continue reading

NanoBiT™ Assay: Transformational Technology for Studying Protein Interactions Named a Top 10 Innovation of 2015

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For three out of the last four years, we have been honored to have one of our key technologies named a Top 10 Innovation by The Scientist. This year the innovative NanoBiT™ Assay (NanoLuc® Binary Technology) received the recognition. NanoBiT™ is a structural complementation reporter based on NanoLuc® Luciferase, a small, bright luciferase derived from the deep sea shrimp Oplophorus gracilirostris.

Using plasmids that encode the NanoBiT complementation reporter, you can make fusion proteins to “report” on protein interactions that you are studying. One of the target proteins is fused to the 18kDa subunit; the other to the 11 amino acid subunit. The NanoBiT™ subunits are stable, exhibiting low self-affinity, but produce an ultra-bright signal upon association. So, if your target proteins interact, the two subunits are brought close enough to each other to associate and produce a luminescent signal. The strong signal and low background associated with a luminescent system, and the small size of the complementation reporter, all help the NanoBiT™ assay overcome the limitations associated with traditional methods for studying protein interactions.

The small size reduces the chances of steric interference with protein interactions. The ultra bright signal, means that even interactions among proteins present in very low amounts can be detected and quantified–without over-expressing large quantities of non-native fusion proteins and potentially disrupting the normal cellular environment. And the NanoBiT™ assay can be performed in real time, in live cells.

The NanoBiT™ assay is already being deployed in laboratories to help advance understanding of fundamental cell biology. You can see how one researcher is already taking full advantage of this innovative technology in the video embedded below:

Visit the Promega web site to see more examples more examples how the NanoBiT™ assay can break through the traditional limitations for studying protein interactions in cells.

You can read the Top 10 article in The Scientist here.

Manipulating Microbiota: A Synthetic Biology Exploration of the Gut

33553646_lMicrobial cells outnumber the cells of our own bodies approximately 10:1, these microbes that live on our skin and along the epithelial linings of our internal tubes make up our microbiota*, and they can have major effects on our health. Most of our microbiota are commensal organisms, living in harmony with our body, but if you suppress our immune system or greatly reduce their populations with large doses of antibiotics, and you will soon see the effects of disrupting our microbiota.

There is much interest in the microbiota that inhabit our bodies. For instance several studies have indicated that intestinal microbes can play a big part in obesity, with changes in the makeup of the microbiota being a major risk factor (1). But many of these organisms are hard to learn about—the ones that inhabit the deep folds of our gut thrive in moist, warm, anaerobic conditions with lots of specialized nutrients, conditions that are very hard to replicate in the laboratory. For that reason, we don’t know much about many of the microbes that are the most abundant within us.

The Human Microbiome Project begun in 2008 by the National Institutes of Health (2) seeks to understand human microbiota and their relationship to human health. To do this, the researchers leading the project took a metagenomic approach—using advanced DNA sequencing technologies to sequence the genomes of human microbiota and get a look at the human microbiome—without culturing the microbes.

But to truly understand their biology, and to perhaps exploit what we learn to enhance human health we need to be able to manipulate these organisms. In particular, biologists who are interested in synthetic biology would like to use these micro-organisms to monitor what is going on in our bodies, particularly our guts. What better monitor for these hard-to-access places than an organism that is already well adapted to live there?  Continue reading

If We Could But Peek Inside the Cell …Quantifying, Characterizing and Visualizing Protein:Protein  Interactions

14231183 WB MS Protein Interactions Hero Image 600x214Robert Hooke first coined the term “cell” after observing  plant cell walls through a light microscope—little empty chambers, fixed in time and space. However,  cells are anything but fixed.

Cells are dynamic: continually responding to a shifting context of time, environment, and signals from within and without. Interactions between the macromolecules within cells, including proteins, are ever changing—with complexes forming, breaking up, and reforming in new ways. These interactions provide a temporal and special framework for the work of the cell, controlling gene expression, protein production, growth, cell division and cell death.

Visualizing and measuring these fluid interactions at the level of the cell without perturbing them is the goal of every cell biologist.

A recent article by Thomas Machleidt et al. published in ACS Chemical Biology, describes a new technology that brings us closer to being able to realize that goal. Continue reading

Targeting MYC: The Need to Study Protein:Protein Interactions in Cells

Crystal Structure of MYC MAX Heterodimer bound to DNA ImageSource=RCSB PDB; StructureID=1nkp; DOI=http://dx.doi.org/10.2210/pdb1nkp/pdb;

Crystal Structure of MYC MAX Heterodimer bound to DNA ImageSource=RCSB PDB; StructureID=1nkp; DOI=http://dx.doi.org/10.2210/pdb1nkp/pdb;

In 1982, picked up because of its homology to chicken virus genes that could transform cells, MYC became one of the first human genes identified that could drive cellular transformation (1,2). Since that time countless laboratories have prodded and poked the human MYC gene, the MYC protein, their homologs in other animal models, and their transforming viral counterparts.

MYC is a transcription factor and forms heterodimers with a required protein partner, MAX, before binding to the E box sequences of DNA regulatory regions (3). MYC regulates gene expression of many targets through interactions with a host of proteins, often referred to as the MYC Interactome (2).  In fact, MYC is estimated to bind 10–15% of the genome, and it regulates the expression of genes that  are transcribed by by each of the three RNA polymerases (2).

MYC plays a central role in regulating cell growth, proliferation, apoptosis, differentiation and transformation, acting as a central integrator of cellular signals. MYC is tightly regulated at multiple levels from gene expression to protein stability. Dysregulation (usually upregulation) of the amount and stability of Myc protein is observed in many human cancers. Even in cancers in which MYC is not directly involved in transforming cells, its normal expression is often required to support the extracellular matrix and/or vascularization necessary for tumor growth and formation (4).

Because MYC is such a central player cancer pathology, it is an attractive target for cancer therapeutics  (2) . Continue reading