Synthetic Biology by the Letters

Synthetic biology has been in the news a lot lately—or maybe it only seems like it because I’m usually thinking about our partnership with the iGEM Foundation, which is dedicated to the advancement of synthetic biology. As the 2019 iGEM teams are forming, figuring out what their projects will be and how to fund them, it seemed fitting to share some of these stories.

A, C, T, G…S, P, Z, B?

Researchers recently developed four synthetic nucleotides that, when combined with the four natural nucleotides (A, C, T and G), make up a new eight-letter synthetic system called “hachimoji” DNA. The synthetic nucleotides—S, P, Z and B— function like natural DNA by pairing predictably and evolving.

While this development may seem superfluous, there are a handful of useful applications for extra letters in the DNA alphabet, such as DNA data storage. These applications hinge on the fact that doubling the number of nucleotides increases the number of possible codons from just 64 to 4,096.

The next step for scientists is developing the other molecular components necessary to read hachimoji and create synthetic proteins with novel properties and functions. Currently, humans make 20 amino acids; additional codons could mean the possibility of new amino acids and proteins with valuable characteristics.

ABEs and CBEs

While CRISPR-Cas9 always seems to be in the news, it has received a lot of recent criticism for its potential lack of specificity. Base editing is a technique that uses components of the CRISPR-Cas9 system to target specific nucleotides within a genome for point mutations, offering more precise and efficient gene editing.

Traditional CRISPR-Cas9 uses a guide RNA to target a specific gene sequence and recruit Cas9 nuclease to cut the DNA. This can be used to knock out a gene via indels created during repair or a desired sequence change can be introduced using a DNA template and homology-driven repair. Although indels usually result in conversion of greater than 50% of alleles, homology-driven repair has an efficiency of less than 1%.

Base editing uses Cas9 proteins modified to lack nuclease activity. These proteins are fused to enzyme domains that facilitate conversion of cytosines to thymines or adenines to guanines. Cytosine base editors (CBEs) convert C-G base pairs to T-A and adenine base editors (ABEs) convert A-T base pairs to G-C. (Check out this infographic of ABEs and CBEs.)

Guide RNAs then direct the CBE or ABE to a site of interest within the genome, which results in more precise editing by swapping out C-G base pairs for A-T or vice versa. Although it has comparable efficiency (ranging from 5-50%) to traditional CRISPR-Cas9, the real benefit to base editing is that it reduces the likelihood of off-target mutations since it doesn’t cut DNA.

This technique was originally developed for us in mammalian cells, but it was recently adapted for plants. This lab applied the technique by converting Arabidopsis thaliana to a late-flowering variety by base editing the gene for a single amino acid. Another lab created albino plants by altering a single splice site. This is an important development that expands the available toolkit for editing plant genomes.

THC and CBD

Speaking of plants, Cannabis has also been in the news a lot lately—particularly one of its constituent compounds, CBD, which is being added to everything from beer to lip balm. Purifying cannabinoids like CBD or THC from all of the other plant products present can be challenging. By using microbes to produce these compounds through fermentation, the problem of contamination is eliminated.

A recent paper in Nature explains how one lab did just that. The lab modified yeast by introducing several genes for enzymes that convert galactose into CBGA, a cannabinoid. The yeast had also been transformed to contain genes that could make inactivated forms of either THC or CBD. Once the yeast had made their respective cannabinoid, they could be activated by heating the yeast.

This work is a proof-of-concept that the introduced genes can accomplish cannabinoid synthesis in a single yeast cell. To compete with plant-based production of cannabinoids, the yeast output would need to be scaled up significantly. In addition, this technique could be used to synthesize novel cannabinoids, which could be studied further and might lead to new medicines.

I Spy DNA

Biohackers have created a way to spy on synthetic DNA machines. They were able to carry out what they call an “acoustic side-channel attack” on common DNA synthesis instrument. By using an audio recorder, the researchers listened to the noise made by the machine while it went through the process of directing chemical reactions to assemble DNA sequences.

The unique sounds of the moving parts of the process—tubes, valves, liquids—moving in the machine were analyzed using machine learning models to identify what it “sounds” like when each nucleotide (A, G, T or C) is add to the sequence. While this approach isn’t something you would expect to occur outside of a research lab right now, it could be on the horizon.

Devices like voice assistants in labs with automated processes could be hacked to listen in and steal information. This could pave the way for stealing proprietary sequences related to novel synthetic biology. Looking to the future, this could also complicate the efforts to use DNA for data storage since the possibility of this kind of attack would raise security concerns.

Perhaps these feats of synthetic biology (or so many others I didn’t include) will inspire iGEM teams and other young scientists, setting them on a path to discovering other breakthroughs in the field.

We’re curious about what other synthetic biology discoveries are inspiring you—please let us know!

From BTCI to Africa and Back Again: One Student’s Journey in Science Education

Today’s blog is brought to us by and alumus of Dane County Youth Apprenticeship Program, Aidan Holmes.

In this blog I have the opportunity to write about how my experiences at the BTC Institute as a high school student were instrumental in leading me to my passion for science education, my Peace Corps experience, and my current role as a biotechnology instructor for the very same institute.

I became familiar with the BTC Institute as a student at Marshall High School when our biology teacher organized a biotechnology field trip for us. I loved learning about DNA and biotechnology since 7th grade so attending a field trip like this was an incredible opportunity to engage in hands-on biotechnology. When I learned about the Youth Apprenticeship Program in Biotechnology I knew I had to apply and enrolled during my senior year of high school. Through the program I took a weekly class at the BTC Institute and I worked as a student researcher in a biochemistry lab at UW-Madison. I enrolled for classes at UW-Madison the following year and pursued an undergraduate degree in genetics and a certificate in education and educational services. Continue reading “From BTCI to Africa and Back Again: One Student’s Journey in Science Education”

Mutation Analysis Using HaloTag Fusion Proteins

In a recent reference, Kinoshita and colleagues characterized the phosphorylation dynamics of MEK1 in human cells by using the phosphate affinity electrophoresis technique, Phos-tag sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Phos-tag SDS-PAGE; 1). They found that multiple variants of MEK1 with diferent phosphorylation states are constitutively present in typical human cells.

To investigate the relationships between kinase activity and drug efficacy researchers from the same laboratory group conducted phosphorylation profling of various MEK1 mutants by using Phos-tag SDS- PAGE (2).

They introduced mutations in of the MEK-1 coding gene that are associated with spontaneous melanoma, lung cancer, gastric cancer, colon cancer and ovarian cancer were introduced into Flexi HaloTag clone pFN21AE0668, which is suitable for expression of N-terminal HaloTag-fused MEK1 in mammalian cells. Continue reading “Mutation Analysis Using HaloTag Fusion Proteins”

Cardboard Couture: From Conception to Runway Debut

The five-member team at the Read(y) To Wear event.

What do fashion, paperboard product packaging and literacy have in common? Answer: The Read(y) to Wear submission from a team of Promega employees for an event put on by the Madison Reading Project. With a challenge that stated teams need to make a garment mostly of paper, the resulting creations would be displayed on a runway as part of a charitable evening for an organization dedicated to bringing books to children.

Volunteering to be part of what became a five-person team to create a wearable garment from paper was the easy part. Our first few meetings we were experimenting with ideas and techniques using paper we could access on campus: Print catalogs, discarded books and our prototype product kit boxes. It was the kit boxes with the David Goodsell imagery that inspired our ideas to create a suit of armor. The paperboard boxes protect the products we ship to customers like a suit of armor protects warriors in battle. Continue reading “Cardboard Couture: From Conception to Runway Debut”

Science Visitors Only: Watching Life Grow on a New Island

We spend a lot of time looking at history and imagining—”what was it like when…?” As a biologist, I find myself most drawn to stories about the evolution of life. Why does this plant have purplish leaves? How did this species end up in a symbiotic relationship with this other species? How did this animal get to this tiny island 20 miles off the Southern coast of Iceland?

The volcanic island of Surtsey erupting in 1963.
The newly formed island of Surtsey erupting in 1963.

That last one was too specific to be rhetorical, wasn’t it? The volcanic island of Surtsey broke the ocean surface on November 14, 1963, and continued to erupt until June 5, 1967, reaching its maximum size of 2.7 km2 (about the size of Central Park in New York City). At this size, it was large enough to be a good site for biocolonization. Only a few scientists are allowed to visit the island, ensuring that colonization of the island can occur without human interference. Continue reading “Science Visitors Only: Watching Life Grow on a New Island”

Colorectal Cancer Awareness Making March About More Than Basketball

In the United States, March means college basketball. “March Madness” brings us the excitement and entertainment of the NCAA college basketball championship tournament. But for a dedicated group of advocates, researchers, patients and families, it means something else entirely. March is colorectal cancer awareness month.

 

According to the American Cancer Society, colorectal cancer will be the third most frequently diagnosed and the second most deadly cancer in the United States in 2019 (1). Most of those who develop colorectal cancer do not have a family history or genetic connection to the disease. However, in some families, cancer occurs more often than expected. A family history of colorectal cancer can suggest a genetic factor. Continue reading “Colorectal Cancer Awareness Making March About More Than Basketball”

A BiT or BRET, Which is Better?

Now that Promega is expanding its offerings of options for examining live-cell protein interactions or quantitation at endogenous protein expression levels, we in Technical Services are getting the question about which option is better. The answer is, as with many assays… it depends! First let’s talk about what are the NanoBiT and NanoBRET technologies, and then we will provide some similarities and differences to help you choose the assay that best suits your individual needs. Continue reading “A BiT or BRET, Which is Better?”

Radical Eradication: A (Population) Crash Course in Genetic Engineering

Malaria is a life-threatening blood disease that plagues nearly two-thirds of the world’s population. The disease in manifested by parasites of the Plasmodium genus and transmitted to humans through the bite of female Anopheles mosquitoes, which serve as the primary disease vectors. Roughly 200 million people per year are infected with malaria, and approximately 400,000 deaths are reported annually, with children under the age of five comprising the majority of victims.

Africa disproportionately bears the global brunt of this devastating illness, with approximately 92% of all reported cases, as well as 93% of all reported deaths, originating from the continent. This can be partially attributed to the fact that the conditions for transmission are essentially ideal there: the principal vector species Anopheles gambiae are abundant in this region, and not only do they prefer to source their blood from humans over animals, but the mosquitoes also tend to have a longer lifespan, which allows the most common and deadly malaria parasite, Plasmodium falciparum, to complete its life cycle, which contributes to higher disease transmission efficacy.

Though malaria is a preventable disease, often the areas affected most lack access or resources, or are politically unstable, all factors that can contribute to the absence of consistent, functional malaria control programs. Though malaria is also a curable disease, it has long been debated whether eradication was even within the realm of possibility. There are four species of Plasmodium parasites responsible for the pathogenesis of malaria and each exhibit different forms of drug resistance and each responds differently to different medications. This alone makes the prospect of developing a single overarching vaccine for all strains of malaria an improbable achievement and the idea of eradication practically impossible.

A CRISP[E]R APPROACH

In a study recently published in Nature Biotechnology, a team of scientists were able to effectively implement a new, though indubitably controversial, type of genetic modification. The team was able to weaponize mosquitoes to take out…other mosquitoes! They were able to engineer male mosquitoes to rapidly pass down a fatal mutation through generations of their own species, effectively sterilizing all female offspring, eliminating the possibility of successful reproduction and resulting in a population crash. Continue reading “Radical Eradication: A (Population) Crash Course in Genetic Engineering”

Voted Drug Discovery and Development Product for 2019: NanoBRET TE Kinase Assays

Choice Drug Discovery and Development Product 2019 award
Michael Curtin, Promega, accepting the Reviewers’ Choice for Drug Discovery and Development Product of the Year award from SelectScience.

As announced at SLAS in Washington, D.C. recently, we are excited to have NanoBRET Target Engagement (TE) Intracellular Kinase Assays awarded the SelectScience Reviewers’ Choice for Drug Discovery and Development Product of the Year 2019!

The NanoBRET™ Target Engagement (TE) Kinase Assay, first available in the fall of 2017, has been getting great reviews on the SelectScience site for more than a year now. Continue reading “Voted Drug Discovery and Development Product for 2019: NanoBRET TE Kinase Assays”

The Secret Fluorescent Life of Flying Squirrels

flying squirrel specimen
A flying squirrel museum specimen under normal light versus ultraviolet light. Photo courtesy of AM Kohler, et al.

In May 2017, a surprising discovery was made in the woods of Bayfield County, Wisconsin, just about a 5-hour drive north of Promega headquarters. Jonathan Martin, Associate Professor of Forestry at Northland College, was exploring the forest with an ultraviolet (UV) light in search of fluorescent lichen or plant life. What he found instead was a bright pink glow coming from a most unexpected source—a flying squirrel.

Continue reading “The Secret Fluorescent Life of Flying Squirrels”