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Site Directed Mutagenesis by PCR

Posted by Guest Blogger on Aug 2, 2016 10:30:00 AM

This post was contributed by guest blogger, Kristian Laursen from Cornell University.

Site directed mutagenesis is a highly versatile technique that can be used to introduce specific nucleotide substitutions (or deletions) in a tailored manner. The approach can be used in conventional cloning (to introduce or remove restriction sites), in mapping of regulatory elements (to mutate promoters/enhancers in reporter constructs), in functional analysis of proteins (to perform alanine scanning mutagenesis or targeted substitution of key residues), and in SNP analysis (to introduce naturally occuring SNPs in a plasmid context). The technique is also highly relevant in this age of CRISPR; site-directed mutagenesis generally applies to plasmids, but may also facilitate genome editing. Tailored mutations are commonly introduced to endogeneous DNA through homology-directed repair (HDR) of a CRISPR/Cas9 induced double-stranded break. This site-directed genome editing requires a template of high homology to the endogenous target, yet to facilitate the repair, the template should be resistant to Cas9 cleavage. If a plasmid contains the template, site-directed mutagenesis can be used to mutate the PAM sequence (an NGG sequence critical for Cas9 cleavage), thereby rendering the resulting construct resistant to Cas9 induced cleavage.

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Topics: Lab Tips, Protocols

Recombinase-based State Machines Enable Order-dependent Logic in vivo

Posted by Guest Blogger on Jul 28, 2016 10:30:00 AM

This post was contributed by guest blogger Nathaniel Roquet, a PhD student in the Harvard Biophysics program and researcher in the Lu Lab at MIT.

Note: The following blog post reduces the content of our paper, “Synthetic recombinase-based state machines in living cells” (1), into a more straight-forward, concise explanation of how to adapt our engineered devices, recombinase-based state machines for your own experimental needs. For more context, exposition, and detail, please refer to the paper.

Why Might One Be Interested in State Machine Technology?

Biological research has produced a massive amount of information regarding which regulatory proteins, signaling molecules, mutations, and environmental conditions drive certain cellular behaviors, but little is known about the order or timing of these factors. Recombinase-based state machines (RSMs), which take on a particular DNA-sequence configuration (state) based on the identity and order of a particular set of inputs, may be used to better understand and engineer cellular processes that are influenced by temporally ordered biochemical events.

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Topics: Genome Engineering, Synthetic Biology

AAV: A Versatile Viral Tool for Gene Expression in Mammals

Posted by Guest Blogger on Jul 26, 2016 10:30:00 AM

This post was contributed by guest blogger Didem Goz Ayturk, a Postdoctoral Fellow in Connie Cepko’s Lab at Harvard Medical School with edits from Addgenie Karen Guerin.

Adeno-associated virus (AAV) has emerged as a favorite viral tool for both research and clinical applications. AAV can be used to transiently express a gene of interest in a variety of cell types. It was first described about 50 years ago as a contaminant of adenoviral preparations, hence the name (Atchison et al., 1965) AAV is a single stranded, DNA virus belonging to the family Parvoviridae. It has a "simple" genome packaged in an icosahedral capsid. It does not have a lipid coat, also called an envelope, and thus cannot support the addition of a glycoprotein, such as VSV-G, to its surface. In research applications, the genome is typically gutted so that precious cargo space is opened for gene delivery, and for safety. You can easily complement the virus in a tissue culture setting, in other words “in trans”, by supplying the genes that encode the replicase functions and capsid proteins. This gives researchers the ability to produce more virus in a controlled setting. Even though AAV is isolated from a wide range of organisms, it has not been associated with disease, and it is considered a biosafety level 1 (BSL1) viral agent.

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Topics: Viral Vectors

Gendered Innovations: Why Does Sex of the Cell Matter?

Posted by Guest Blogger on Jul 21, 2016 10:30:00 AM

The post was contributed by guest blogger Londa Schiebinger, PhD, Hinds Professor of History of Science, Stanford University.

Sex and gender are critical components of biological research that are often forgotten or ignored. If we wish to conduct research that fails less and helps more people, we need to take sex into account. Gendered Innovations is an international, collaborative project—funded by the European Commission, the US National Science Foundation, and Stanford University—that harnesses the creative power of sex and gender analysis for innovation and discovery.

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Topics: Lab Tips

Special Delivery: Fluorophore Targeting for FRET Studies

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

This post was contributed by guest blogger James D. Fessenden, an Assistant Professor at Brigham and Women’s Hospital.

Biochemists often struggle to understand how a protein of interest actually behaves. How large is it? What parts of it move when you feed it substrate or add an essential cofactor? How many binding partners does it have and how do they come off and on in a cellular environment? If these are pressing issues in your laboratory, then FRET experiments are a viable biophysical path to answers.

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Topics: Lab Tips, Fluorescent Proteins

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