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When is a Monomer not a Monomer? The Top Three Ways Your Favorite Fluorescent Protein Oligomerizes in Cells

Posted by Guest Blogger on Apr 19, 2016 10:30:00 AM

This post was contributed by guest blogger Erik L. Snapp.

Stop using EGFP/GFP for fusion proteins! Despite multiple studies in high profile journal articles, many researchers remain unaware that EGFP/GFP is prone to forming noncovalent dimers. This property of EGFP can lead to significant artifacts.

If you're using green fluorescent protein or Enhanced Green Fluorescent Protein (GFP/EGFP) for a transcriptional reporter or as a general cytoplasmic label of cells, there's no problem. You're OK. However, if you fuse your protein of interest (POI) to GFP to study the protein's behavior in cells, in solution or something in between, you are using a tag with a serious drawback. The standard EGFP plasmid that used to be sold by Clontech and is in a freezer box in just about every lab in the world, is not inert. In all seriousness, EGFP/GFP has a real nontrivial propensity to noncovalently dimerize. That means that your POI fused to GFP or another fluorescent protein (FP) could be forming dimers in cells. Why should you care? Three simple ways a dimeric FP could ruin your day (and experiment) are listed below. Solutions to avoid these all too common issues follow.

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Topics: Imaging, Fluorescent Proteins

R Bodies: Membrane-Rupturing Microscopic Tools

Posted by Guest Blogger on Apr 14, 2016 10:30:00 AM

This post was contributed by guest blogger Jessica Polka, a Postdoctoral Research Fellow with Pamela Silver. 

Most types of biological motion (whether endocytosis, vesicle trafficking, or muscle contractions) are produced by orchestrated movements of networks of proteins consuming molecular fuel sources. While the importance of understanding these complex processes can’t be overstated, we can also learn a lot from Nature’s simpler solutions to transmitting forces over long distances. For instance, how much force can be generated by conformational changes in proteins? How can information propagate through a structured material over a long distance? And can we understand such a structure well enough to engineer it to suit our purposes?

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Topics: Plasmid Technology, Synthetic Biology

Pairing CombiGEM and CRISPR for Combinatorial Genetic Screening

Posted by Guest Blogger on Apr 12, 2016 10:30:00 AM

This post was contributed by guest blogger Alan Wong.

The complexity of biological systems can hinder our attempts to study and engineer them, but what if we had a simple tool that allowed us to rapidly decode the complexity? The CombiGEM-CRISPR technology was developed with the goal of providing an easy-to-use tool to analyze the complex combinatorial genetic networks underlying your favorite biological phenotype in a scalable way. This blog post will introduce you to this new technology, and guide you through the basics of CombiGEM-CRISPR experiments.

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Topics: Plasmid Technology, Genome Engineering, CRISPR

Optimizing Donor DNA for Enhanced CRISPR Genome Editing

Posted by Guest Blogger on Mar 24, 2016 10:30:00 AM

This post was contributed by guest blogger Chris Richardson, a Postdoctoral Researcher in Jacob Corn’s lab.

CRISPR-Cas9 (Cas9) is an RNA-guided nuclease that targets and cuts genomic DNA. The interplay between Cas9 (which causes the breaks) and host cell DNA repair factors (which repair those breaks) makes Cas9 extremely effective as a genome editing reagent. This interplay falls into two broad categories and thus, causes two types of editing outcomes: Cas9 breaks repaired by the non-homologous end-joining (NHEJ) pathway disrupt target gene sequences (thus inactivating genes), while breaks repaired by homology directed repair (HDR) pathways can modify the sequence of a gene (thus altering its function). HDR is crucial for certain applications, for example, correcting the allele that causes sickle cell anemia. However, HDR occurs much less frequently than NHEJ and the efficiency of these editing reactions is low. Understanding the biological cause of this repair bias is a fascinating (and yet unanswered) question. Our recent paper (Richardson et al 2016) revealed some of the biophysical parameters that can influence the HDR/NHEJ decision.

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Topics: Genome Engineering, CRISPR

Minigenomes - a Safe Way to Study Dangerous Viruses

Posted by Guest Blogger on Mar 17, 2016 10:30:00 AM

This post was contributed by guest blogger Tessa Cressey.

The highly pathogenic Ebola virus belongs to the group of nonsegmented negative sense RNA viruses, along with other viruses that cause disease in humans such as measles, mumps, and rabies. Research on Ebola virus has been limited, in part, due to the necessity for working with this virus under the highest biosafety level conditions, BSL-4. In this regard, minigenome systems, such as the one developed for Zaire ebolavirus (EBOV) are extremely useful, allowing researchers to study aspects of the EBOV replication cycle under BSL-2 conditions (4).

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Topics: Viral Vectors

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