On-site, In-house Environmental Monitoring to Obtain Species-Level Microbial Identification

Loss of life and serious illness from contamination of manufactured products that are consumed as food or used in medical procedures illustrate the need to prevent contamination events rather than merely detect them after the fact.  High-profile news stories have described contamination events in compounding pharmacies (1), food processing and packaging plants (2) and medical device manufacturers (3). Although contamination in manufacturing settings can be physical, chemical, or biological, this article will focus environmental monitoring to determine the quality of a manufacturing facility with respect to microbial contamination.

Scientist in pharmaceutical manufacturing facility  performing environmental monitoring.

To ensure that the products they produce and package are manufactured in a high-quality, contaminant-free environment, many industries are required to establish routine environmental monitoring programs. Samples are collected from all potential sources of contamination in the production environment including air, surfaces, water supplies and people. Routine monitoring is essential to detect trends such as increases in potential pathogens over time or the appearance of new species that have not been seen before so that contamination events can be prevented.

Because environmental monitoring requires identification to the level of the species, most environmental monitoring programs will collect samples and then send them off to a facility to be sequenced for genomic identification of any microbial species. Such genotypic analysis involves DNA sequencing of ribosomal RNA (rRNA) genes to determine the taxonomic classification of bacteria and fungi. In this method, informative sections of the rRNA genes are amplified by PCR; the PCR products sequenced; the sequence is compared to reference libraries; and the results interpreted to make a species-level identification for a given microbial isolate.

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2023 Promega iGEM Grant Winners: Tackling Global Problems with Synthetic Biology Solutions

On June 15, 2023, we announced the winners of the 2023 Promega iGEM grant. Sixty-five teams submitted applications prior to the deadline with projects ranging from creating a biosensor to detect water pollution to solving limitations for CAR-T therapy in solid tumors. The teams are asking tough questions and providing thoughtful answers as they work to tackle global problems with synthetic biology solutions. Unfortunately, we could only award nine grants. Below are summaries of the problems this year’s Promega grant winners are addressing.

UCSC iGEM

An immature night heron against the green surface of Pinto Lake. 2023 Promega iGEM Grant Winner, UCSC iGEM seeks to mitigate these harmful aglal blooms.
A night heron hunts on Pinto Lake, California.

The UCSC iGEM team from the University of California–Santa Cruz is seeking a solution to mitigate the harmful algal blooms caused by Microcystis aeruginosa in Pinto Lake, which is located in the center of a disadvantaged community and is a water source for crop irrigation. By engineering an organism to produce microcystin degrading enzymes found in certain Sphingopyxis bacteria, the goal is to reduce microcystin toxin levels in the water. The project involves isolating the genes of interest, testing their efficacy in E. coli, evaluating enzyme production and product degradation, and ultimately transforming all three genes into a single organism. The approach of in-situ enzyme production offers a potential solution without introducing modified organisms into the environment, as the enzymes naturally degrade over time.

IISc-Bengaluru

Endometriosis is a condition that affects roughly 190 million (10%) women of reproductive age worldwide. Currently, there is no treatment for endometriosis except surgery and hormonal therapy, and both approaches have limitations. The IISc-Bengaluru team at the Indian Institute of Science, Bengaluru, India, received 2023 Promega iGEM grant support to investigate the inflammatory nature of endometriosis by targeting IL-8 (interleukin-8) a cytokine. Research by other groups has snow that targeting IL-8 can reduce endometriotic tissue. This team will be attempting to create an mRNA vaccine to introduce mRNA for antibody against IL-8 into affected tissue. The team is devising a new delivery mechanism using aptides to maximize the delivery of the vaccine to the affected tissues.

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Conversations: Nerve-Tumor Crosstalk in the Tumor Microenvironment

Cancer cells are characterized by features such as metabolic reprogramming and uncontrolled proliferation all of which are supported by underlying genomic instability, inflammation and the tumor microenvironment.

Cancer cells can be distinguished from normal cells by a variety of features including their ability to inappropriately activate signals for cell proliferation, evade growth suppression from contact inhibition or tumor suppressor activity, evade cell death signals, replicate DNA continually, induce angiogenesis, invade new tissues, reprogram their metabolism to provide energy for rapid proliferation, and evade immune detection (1) . Several biological processes are responsible for these features including genomic instability, inflammation, and the creation of a tumor microenvironment.

The tumor microenvironment is the network of non-malignant cells, connective tissue and blood vessels that surround and infiltrate the tumor. These surrounding “normal” cells interact with each other and the cancer cells and are important players in tumorigenesis. One cell type that is often found in the tumor microenvironment are nerve cells. In fact, cancer cells often express proteins that encourage nerve growth into the tumor microenvironment such as growth factors and axon-guidance molecules (2). Crosstalk between nerve cells and tumor cells can facilitate development of several cancer types (2) including pancreatic, head and neck, oral, prostate, and colorectal cancers.

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Avoid the Cloning Blues This Season

I was blasting a holiday music playlist while driving recently, and Presley’s Blue Christmas played. I couldn’t get the phrase “Christmas Cloning Blues” out of my mind, and by the time I arrived at my destination, this happened:

Cloning Blues Christmas

(to the tune of Blue Christmas by Elvis Presley)

Blue and white colonies on a selective plate. Careful planning can help you avoid the cloning blues
Blue/White cloning is a standard technique in molecular biology labs.

I’ll have a blue Christmas without you

Colonies so blue, insert without you

Incubating my plates at 37 degrees

Won’t be the same if you’re not in lacZ


And all those blue colonies are forming

When my lab mates’ clonings are performing

They’ll be doing alright,

With their plates all filled with white

But I’ll have a blue, blue, blue cloning

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A Virus Makes a Comeback: Emerging Poliovirus Transmission in the West

In 1921, at age 39, Franklin D. Roosevelt, the man who would later be elected the 32nd president of the United States, was diagnosed with polio (poliomyelitis). His symptoms included fever, gastrointestinal issues, numbness, and leg and facial paralysis. The disease left him paralyzed from the waist down, relying on a wheelchair and leg braces to walk.

The paralysis from poliovirus infection affected the involuntary muscles that allow breathing, and iron lungs were used to keep patients breathing until they cleared the infection.
The paralysis from poliovirus infection affected the involuntary muscles that allow breathing, and iron lungs were used to keep patients breathing until they cleared the infection.

At the height of the polio epidemic in 1952, more than 3,000 people died of polio in the United States, and 20,000 more people suffered paralysis. Pictures of the era show children in special hospital wards, inside ominous-looking iron lungs, while “recovered” children played on the grounds of hospitals wearing leg braces.

In 1938, Roosevelt founded the March of Dimes, which funded the development of the Salk polio vaccine. Two years after the introduction of the Salk vaccine in 1955, polio cases in the US dropped by 90%. In fact, sustained polio transmission has been absent from the US for nearly 40 years; according to the CDC, the last case of wild poliovirus in the US occurred in 1979.

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Keeping Cool When It Hurts: An Engineered Alternative to Pharmacological Pain Management

Pain management, particularly long-term pain management, is a difficult problem. Opioids are effective at reducing pain, but they are addictive and carry with them the significant costs of addiction and overdose that affect not only the individual but society as well. 

Saltatory conduction in neurons.  Localized cooling of neurons may provide a non-addictive method for pain management.

Recently, work presented in Science by Reeder and colleagues proposes an engineering solution to pain management: miniature, implantable devices that can deliver localized and reversible neural blocking to reduce pain. While such devices could deliver any kind of stimulus ranging from electrical to pharmacological, the researchers developed a device that temporarily blocks neural signals by cooling.

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Designing Better Therapeutic mAbs: An Assay for Rapid, Parallel Screening of Fc/ FcɣR Interactions

The first monoclonal antibody (mAb) was produced in a lab 1975, and the first therapeutic mAb was introduced in the United States to prevent kidney transplant rejection in 1986. The first mAb used in cancer treatment the anti-CD20 mAb, rituximab, was used to treat non-Hodgkin’s lymphoma and chronic lymphocytic leukemia. Today therapeutic mAbs have become a mainstay of cancer, autoimmune disease, and metabolic disease therapies and include HERCEPTIN® used to treat certain forms of breast cancer, Prolia used to treat bone loss in post-menopausal women, and Stelara used to treat autoimmune diseases like psoriatic arthritis and severe Crohn disease, among many others. Therapeutic mAbs bind targets with high specificity and affinity and they can recruit effector cells to drive target elimination through mechanisms such as antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), making them highly specific, effective therapies.

3D rendering of a Lumit Assay which can be used  for plate-based screening assay to measure the affinities of Fc interactions of therapeutic mAbs.
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Use of ProAlanase Digestion Increases Number of Identified Methylglyoxal (MGO)-Modified Proteins in Whole-Cell Lysates

space filling structural model methylglyoxal (MGO)
Methylglyoxal is responsible for post translational protein modifications, that result in advanced glycation endproducts (AGEs), which are associated with aging and disease.

Post-translational modifications (PTM) of proteins are essential for the function of many proteins, but aberrant modification of protein residues also can interfere with protein function. PTMs occur in two ways. Proteins may be modified through the activity of enzymes such as kinases, phosphorylases, glycosylases and others that add or remove specific chemical moieties to amino acid residues. PTMs can also result from non-enzymatic reaction between electrophilic compounds and nucleophilic arginine and lysine residues within a protein. Metabolites and metabolic by products produced during glycolysis, especially glyoxal and methylglyoxal (MGO), are examples of such electrophilic compounds. These compounds can react with arginine and lysine to produce advanced glycation end products (AGEs), which are biomarkers associated with aging and degenerative diseases such as Alzheimer disease, diabetes and others. MGO is also elevated in tumors that have switched from oxidative phosphorylation to glycolysis as their main energy production pathway.

Only limited information is available about site-specific MGO PTMs in mammal cells, and most studies have focused on measuring the amount of MGO modifications in a treatment scenario compared to a control. Donnellan and colleagues recently published work to identify specific MGO protein modifications.  They used a “bottom-up” proteomic analysis of WIL2-NS B lymphoblastoid whole-cell lysates to identify specific MGO-modified proteins. In particular, the group was looking for modifications in proteins that might explain how MGO activity contributes to aneuploidy.

For the study, 100µg of cellular protein extract was reduced with dithiothreitol and then alkylated with chloroacetamide. The sample was diluted to reduce urea concentration. Trypsin Gold was added and samples were digested for 8 hours at 37°C. Digestion was terminated by adding formic acid. For ProAlanase digestion, 20µg of protein was reduced, alkylated and diluted to reduce urea concentration before adding digesting with ProAlanase for 4 hours at 37°C.

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The authors identified 519 MGO-modified proteins.  Most of the modifications were identified in the trypsin digestion reactions; however, ProAlanase digestion increased the number of MGO modifications identified by approximately 25% (with less than 4% of the modification sites being detected in both the ProAlanase and trypsin digestion reactions. The authors suggest that ProAlanase increased sequence coverage to reveal sites not detected in the trypsin digestions. Therefore, they conclude that ProAlanase can be used along with trypsin digestion to increase the identification of MGO modifications.

ProAlanase can be used along with trypsin digestion to increase the identification of MGO modifications.

MGO-modified proteins from the WIL2-NS whole cell lysates included proteins involved in glycolysis, translation initiation, protein folding, mRNA splicing, cell-to-cell adhesion, heat response, nucleosome assembly, protein SUMOylation and the G2/M cell cycle transition. More work to further characterize the sites of these modifications and their potential effects on the function of the modified proteins is ongoing.


Read more about ProAlanase, a new site-specific endoprotease from Promega.


Literature Cited

Donnelian, L et al. (2022) Proteomic Analysis of Methylglyoxal Modifications Reveals Susceptibility of Glycolytic Enzymes to Dicarbonyl Stress Int. J. Mol. Sci. 23(7), 3689 doi.org/10.3390/ijms23073689

PROTAC Virus Vaccines: A New Approach to Vaccine Development

Vaccine research and development is a major area of focus for life scientists across the globe. Clinical trials have shown that vaccines that target tumors show promise for cancer treatment. Additionally, the emergence of new zoonotic diseases has revealed a need to develop vaccines quickly as the world becomes more global and human populations interact more often with each other and wild habitats. Importantly, these vaccines need to be suitable for distribution in a variety of settings, including those that do not have easy access to refrigeration.

Influenza Virus. Si et al used influenza as a model to engineer and test PROTAC Virus vaccines

There are many ways to classify the different types of vaccines that are currently available. The National Institute of Allergy and Infectious Diseases in the United States, categorizes vaccines as: whole pathogen vaccines, subunit vaccines, and nucleic acid vaccines—based on how the antigen that stimulates the immune response is delivered to the host.

Whole-pathogen vaccines, which include many of vaccines used in clinical settings, use the entire pathogen (organism that causes the disease) that has been either weakened or killed to elicit a protective immune response. Killed vaccines are what their name implies: the pathogen has been killed so that it cannot cause disease, but enough of its structure remains to generate antibody response. Often, the immune response generated with killed vaccines is not as robust as that generated with other kinds of vaccines. 

Weakened or attenuated vaccines use whole pathogens that have been weakened in the laboratory through long-term culture or other means. Our modern MMR (measles, mumps and rubella) vaccine is an example of an attenuated vaccine. These vaccines tend to generate strong, long-lasting immune responses, but have increased risk for immunocompromised individuals.

Engineering an Influenza A PROTAC Virus Vaccine

A recent paper by Si et al published in Nature Biotechnology describes a new type of live-attenuated whole pathogen vaccine: the PROTAC virus. PROTAC viruses are prevented from replicating by targeting critical viral proteins for degradation using the host cell protein degradation pathway. The vaccine is live-attenuated by the host cells that degrade critical proteins.

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Detecting Disulfide Bond Shuffling in Biologics Using Trypsin Platinum

Biologic therapeutics such as monoclonal antibodies and biosimilars are complex proteins that are susceptible to post-translational modifications (PTMs). These chemical modifications can affect the performance and activity of the biologic, potentially resulting in decreased potency and increased immunogenicity. Such modifications include glycosylation, deamidation, oxidation and disulfide bond shuffling. These PTMs can be signs of protein degradation, manufacturing issues or improper storage. Several of these modifications are well characterized, and methods exist for detecting them during biologic manufacture. However, disulfide shuffling is not particularly well characterized for biologics, and no methods exist to easily detect and quantify disulfide bond shuffling in biologics.

Disulfide bond shuffling occurs when the S-S linkage is not between a Cys and its normal partner
Disulfide bonds are important for protein conformation and function

Normally the cysteines in a protein will pair with a predictable or “normal” partner residue either within a polypeptide chain or between two polypeptide chains when they form disulfide bonds. These normal disulfide bonds are important for final protein conformation and stability. Indeed, disulfide bonds are considered an important quality indicator for biologics.

In a recently published study, Coghlan and colleagues designed a semi-automated method for characterizing disulfide bond shuffling on two IgG1 biologics: rituximab (originator drug Rituxan® and biosimilar Acellbia®) and bevacizumab (originator Avastin® and biosimilar Avegra®).

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