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Plasmids 101: Luciferase

Posted by Jason Niehaus on Jun 24, 2014 11:59:00 AM

Luciferases are a class of enzymes capable of catalyzing chemical reactions in living organisms resulting in the emission of photons. The most familiar bioluminescent organism for most people is the firefly (Photinus pyralis) and perhaps not surprisingly it is also the most commonly used bioluminescent reporter. This beetle emits a yellow-green light with a peak emission at 560nm. Shortly after the initial article describing the cloning of firefly luciferase was published in 1985, several studies utilized luciferase as a genetic reporter in plant and mammalian cells. Luciferase assays have since become a gold standard in gene expression analysis and a luciferase gene (one of many available to choose from) is now a common feature in reporter plasmids. 

Learn How Luciferase Can Be Used In Concert with Fluorecent Proteins in Nano Lanterns

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Topics: Plasmid Technology, Plasmids 101

Designer PUF Proteins for Any RNA Target

Posted by Kendall Morgan on Jun 17, 2014 3:57:00 PM

With the meteoric rise of CRISPR technology, the ability to direct enzymes – from nucleases to transcription factors – to specific sequences of DNA has become commonplace. This ability has opened up a world of possibilities in the engineering of complex gene networks. A comparable system for targeting specific sequences of RNA is highly desirable for extending the complexity of genetic circuits, allowing for tighter spatio-temporal control of gene expression within a cell. Thanks to the work of Huimin Zhao and colleagues, we now have just the tool…designer PUF proteins!

A newly available PUF Assembly Kit makes it possible to devise RNA binding proteins to hit any target of interest. The new tool was developed and implemented by applying the Golden Gate cloning method to human proteins known as Pumilio/fem-3 mRNA binding factors (PUF). In a single step, researchers can now assemble designer PUF domains for RNA specificity engineering.

“The RNA binding domain is interesting because by changing certain amino acids you can change the specificity,” explained Zhanar Abil of the University of Illinois at Urbana-Champaign.

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Topics: Plasmid Technology, Synthetic Biology, Plasmid Kits

Interview: Nicola Patron on Plant Synthetic Biology, MoClo, and More

Posted by Kendall Morgan on Jun 12, 2014 11:30:00 AM

Nicola Patron is Head of Synthetic Biology at the Sainsbury Laboratory, where she often feels more like an engineer than a biologist. Their focus at the lab is on plant-pathogen interactions, and her aim is to produce constructs and edit genomes so as to make plants, and agricultural crops in particular, resistant to disease. They also devise biosensors designed to elucidate the molecular interactions that go on between plants and their pathogens.

As Patron explains it, her work has always been focused on gene transfer, from transgenes to plants, chloroplast to the nucleus, or pathogens to their hosts. I spoke with her about what motivates her research, the MoClo Kit she and Sylvestre Marillonnet share with the scientific community via Addgene, the struggles of plant scientists and how they work to overcome them, and why she spends some of her time engaging with others on Twitter, among other things.

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Topics: Plasmid Technology, Scientific Sharing, Synthetic Biology, Plant Biology, Plasmid Kits

Which Fluorescent Protein Should I Use?

Posted by Guest Blogger on May 20, 2014 10:06:00 AM

This post was contributed by Gal Haimovich of greenfluorescentblog.

Be honest.  Do you really know how fluorescent proteins glow?  

Fluorescent Proteins (FPs) were first discovered over 50 years ago, with the discovery of the Green Fluorescent Protein (GFP), a protein from the jellyfish Aequorea Victoria. Since that discovery, the family of FPs just keeps getting larger with hundreds of variants available. Read on to familiarize yourself with the available FP emission colors and 10 points to keep in mind when choosing an FP (or two) for your upcoming experiments.

Fluorescence is the emission of light by a substance that has absorbed light. The emitted light is at a longer wavelength than the exciting wavelength. Thus, FPs are proteins with this unique capacity.

Many of these FPs are fluorescent when ectopically expressed in most organisms. Furthermore, fusing FPs to another protein usually does not affect its fluorescence. Therefore, FPs are used to study many biological questions. The two most common uses are: 1) to test the expression level in a specific system (by measuring the fluorescence intensity); and 2) to visualize the localization of the FP (fused to the protein of interest), thus tracking the localization of that biomolecule inside living cells.

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Topics: Plasmid How To, Plasmid Technology, Fluorescent Proteins

Hot Plasmids: FRET-Based Biosensors

Posted by Kendall Morgan on May 6, 2014 9:07:55 AM

Oliver Griesbeck of the Max Planck Institute for Neurobiology has been working on genetically encoded indicators of calcium and other small molecules since the very beginnings of the field. Those engineered sensors were designed to replace synthetic calcium dyes, which had been in use since the 1980s.

“Synthetic dyes were the standard in the field, but there is one problem: how to get that into the cells of interest,” Griesbeck said. Because they are chemical compounds, they have to be applied or injected, and they don’t always end up where you want them to go.

Griesbeck is motivated by a particular interest in monitoring the activity and biochemistry of living neurons in an effort to understand the connection between molecular- and cellular-level events and behavior. It’s a problem that he considers “one of the greatest challenges of neuroscience.” 

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Topics: Plasmid Technology, Hot Plasmids, Fluorescent Proteins

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