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Mary Gearing

Mary Gearing is a Scientist at Addgene. She got her start as a Science Communications Intern writing for the Addgene blog and website. As a full-time Addgenie, she still enjoys blogging about CRISPR and other cool plasmids!

Recent Posts

Plasmids 101: Sequence and Ligation Independent Cloning (SLIC)

Posted by Mary Gearing on Dec 17, 2015 10:30:00 AM

If cloning methods had personalities, SLIC (sequence- and ligation-independent cloning) would be a true rebel. Not only does this system not use site-specific recombination, it also doesn’t require a ligation step! Based on the robust system of homologous recombination found in E. coli, SLIC is a cheap, standardized, and rapid multi-part DNA assembly method - read on to learn how to use it in your research.

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Topics: Plasmid How To, Plasmids 101, Protocols, Plasmid Cloning

Teaching an Old DOG New Tricks: Controlling Protein Activity with GFP

Posted by Mary Gearing on Nov 24, 2015 10:30:00 AM

At Addgene, we love GFP, and we’re always excited when depositors find new ways to make this workhorse protein even more useful! From FPs optimized for oxidizing environments to photoconvertible variants, it seems like GFP is always learning new things. Now, work from Connie Cepko’s lab allow researchers to activate transcription or Cre recombinase activity only in the presence of GFP. These systems, known as T-DDOG and Cre-DOG, respectively, repurpose popular GFP reporter lines for more sophisticated experimental manipulations, saving the time and money needed to develop new lines.

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Topics: Plasmid Technology, Synthetic Biology, Fluorescent Proteins, Cre-lox

Inntags: Innovative Protein Epitope Tagging

Posted by Mary Gearing on Nov 10, 2015 10:30:00 AM

First described in the 1980s, protein tags are now one of the most useful items in a scientist’s toolbox. As we’ve covered in Plasmids 101, tags can help you determine localization of a protein of interest, purify it, or determine its expression level without the need for a custom antibody. There is one major caveat - a tag may interfere with protein localization and/or function, so each tagged protein must be tested carefully to ensure it retains the attributes of the native protein. Since this process takes a lot of time and energy, Martí Aldea and collaborators have created a set of “innocuous tags” (inntags) less likely to alter a protein’s properties.

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Topics: Hot Plasmids, Plasmid Elements

Cpf1: A New Tool for CRISPR Genome Editing

Posted by Mary Gearing on Oct 14, 2015 10:30:00 AM

This post was updated on Dec 5, 2017.

In 2015, Zetsche et al. added to the CRISPR toolbox with their characterization of two Cpf1 orthologs that display cleavage activity in mammalian cells. Like Cas9 nucleases, Cpf1 family members contain a RuvC-like endonuclease domain, but they lack Cas9’s second HNH endonuclease domain. Cpf1 cleaves DNA in a staggered pattern and requires only one RNA rather than the two (tracrRNA and crRNA) needed by Cas9 for cleavage. In certain cases, Cpf1 may be better suited for genome editing than Cas9 - read on to learn more about Cpf1 and check out our CRISPR guide for a refresher on CRISPR/Cas9. 

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Topics: Genome Engineering, CRISPR

pSiM24: Simplifying Plant Genetic Engineering

Posted by Mary Gearing on Sep 29, 2015 10:30:00 AM

As previous blogs have noted, plants are an important foundation for life on Earth. Selective breeding methods have shaped the plants that we grow and eat, and genetic engineering will continue to improve plant nutrition, yield, and pest resistance. Much of plant genetic engineering revolves around Agrobacterium tumifaciens. Agrobacterium carries a “tumor-inducing” or Ti plasmid, which allows it to transfer genetic material into the host plant genome. Scientists have worked to optimize this system for gene transfer, studying the stability of modified Ti plasmids during plant infection, as well as plasmid yield during preparation in E. coli. Addgene depositor Indu Maiti has created a new and versatile binary Ti vector for both transient and stable gene expression applications in plants. This smaller, easily customizable vector functions in multiple species, including tobacco and Arabidopsis.

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Topics: Plasmid Technology, Plant Biology

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