Deep Mutational Scanning with One Pot Saturation Mutagenesis

Posted by Beth Kenkel on Feb 22, 2017 10:30:00 AM

Scientists use deep mutational scanning to simultaneously test how multiple amino acid changes affect a protein of interest’s function. This technique relies on the generation of a plasmid library that expresses all desired variants of a protein. Applying a selective pressure winnows the pool down to plasmids expressing variants with optimal function. High-throughput DNA sequencing is then used to measure the frequency of each variant during the selection process. Each variant is assigned a functional score based on its library frequency before selection compared to its library frequency after selection. Key to this process is the ability to generate full libraries of mutant proteins. Researchers from the Whitehead lab developed One pot saturation mutagenesis as a quick and easy technique that can be used to generate complex libraries of mutant plasmids ready for deep mutational scanning.

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Topics: Techniques

Lambda Red: A Homologous Recombination-based Technique for Genetic Engineering

Posted by Guest Blogger on Dec 15, 2016 10:57:02 AM

This post was contributed by guest blogger, Beth Kenkel, a research scientist at the University of Washington.

Restriction enzyme cloning is the workhorse of molecular cloning; however, one of its biggest limitations is that sequence modifications can only be made at restriction enzyme cut sites. The lambda red system is an alternative method that can be used for cloning or genome engineering and is based on homologous recombination. It allows for direct modification of DNA within E. coli and is independent of restriction sites. The lambda red system is derived from the lambda red bacteriophage and its use as a genetic engineering tool is frequently called recombineering - short for homologous recombination-mediated genetic engineering.  It can be used to make an assortment of modifications: insertion and deletion of selectable and non-selectable sequences, point mutations or other small base pair changes, and the addition of protein tags. It also has the flexibility to modify the E. coli chromosome, plasmid DNA or BAC DNA. 

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Topics: Genome Engineering, Techniques, Microbiology

Plasmids 101: TOPO Cloning

Posted by Lianna Swanson on Oct 27, 2016 10:30:00 AM

Toposiomerase based cloning (TOPO cloning) is a DNA cloning method that does not use restriction enzymes or ligase, and requires no post-PCR procedures. Sounds easy right? The technique relies on the basic ability of complementary basepairs adenine (A) and thymine (T) to hybridize and form hydrogen bonds. This post focuses on "sticky end" TOPO (also called TOPO-TA) cloning; however, the TOPO cloning technique has also be adapted for blunt end cloning.

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Topics: Plasmid Technology, Plasmids 101, Techniques, Plasmid Cloning

Editor's Choice, September 2016

Posted by Tyler Ford on Oct 7, 2016 12:00:00 PM

Read All of Our Editor's Choice Blog Posts

As I’m sitting in the San Francisco International Airport listening to the Lion King soundtrack and writing this post, it is my pleasure to announce that we once again reached new heights on the Addgene blog: we surpassed 60,000 views for the month of September! Historically we do better in September than in the summer months, but this is also our best month ever! Hats off to all of our wonderful writers and all those who have helped edit over the past couple of months. Read on to discover what new post contributed the most to this record breaking month and to find other posts that deserve a second look.

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Topics: Lab Tips, Techniques, Editor's Choice

Technique: Probe Phage Genomes for Host Binding Proteins

Posted by Guest Blogger on Sep 29, 2016 10:30:00 AM

This post was contributed by guest blogger, Jessica Sacher, a microbiology PhD student at the University of Alberta studying with the Szymanski lab.

Reasons to Study How a Phage Recognizes Its Host

Bacteriophages (viruses that prey on bacteria) may be the most numerous and most diverse biological entities on our planet, but we still know collectively little about how they infect and influence the evolution of their bacterial prey. Currently, receptor binding proteins (RBPs, the host recognition factors of phages) constitute one of the most popular classes of phage proteins to study. These are highly useful for the biotech industry, which is in the process of capitalizing on phage RBPs as diagnostic tools and therapeutics. In addition, the strategic use of whole phages as therapeutics, which is also gaining a lot of new traction lately (1, 2), depends on knowledge of the structure(s) a given phage will recognize on a host cell.

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Topics: Protocols, Techniques, Microbiology

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