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Plasmids 101: Cre-lox

Posted by A Max Juchheim on Jan 13, 2015 10:47:00 AM

In previous posts for our Plasmids 101 series, we examined a number of important plasmid elements – promoters, origins of replication, protein tags, and antibiotic resistance markers (just to name a few). In this edition, we’re going to take a look at a very interesting tool that can be used for creating (excuse the pun) specific, targeted DNA modifications in transgenic animals, embryonic stem cells, and/or tissue-specific cell types: Cre-lox recombination.

What is Cre-lox?

The Cre-lox system is a technology that can be used to induce site-specific recombination events. The system consists of two components derived from the P1 bacteriophage: the Cre recombinase and a loxP recognition site. The P1 bacteriophage uses these components as part of its natural viral lifecycle, and researchers have adapted the components for use in genome manipulation.

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Topics: Plasmid Technology, Plasmid Elements, Plasmids 101

Interview: Hodaka Fujii on enChIP, New CRISPR Tools, and More

Posted by Larissa Haliw on Dec 2, 2014 2:23:00 PM

Hodaka Fujii, M.D., Ph.D., is an Associate Professor at Osaka University. The Fujii lab specializes in developing novel technologies to analyze molecular mechanisms of genome functions such as epigenetic regulation and transcription by using locus-specific chromatin immunoprecipitation (locus-specific ChIP). These methods consist of insertional chromatin immunoprecipitation (iChIP) and engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP), both developed in the lab. In June 2014, Dr. Fujii joined Addgene's Advisory Board. 

Addgene: Your lab has worked extensively with enChIP systems. Can you describe this technology and its advantages?

Fujii: In the last several years, my lab has been working on development of technologies for biochemical analysis of genome functions such as transcription and epigenetic regulation. To elucidate molecular mechanisms of regulation of genome functions, we need to identify molecules associated with specific genomic regions of interest in a non-biased manner. To achieve this goal, it is necessary to isolate specific genomic regions while retaining molecular interactions.

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Topics: Plasmid Technology, Interview, Investigator Feature

Plasmids 101: Multicistronic Vectors

Posted by Melina Fan on Sep 9, 2014 4:20:00 PM

Co-expression of multiple genes is valuable in many experimental settings. To achieve this, scientists use a multitude of techniques including co-transfection of two or more plasmids, the use of multiple or bidirectional promoters, or the creation of bicistronic or multicistronic vectors. Unlike promoters which will create unique mRNA transcripts for each gene that is expressed, multicistronic vectors simultaneously express two or more separate proteins from the same mRNA. We've discussed promoters before so in this blog post we’ll cover basics of multicistronic vectors: why they are useful, how they work, and how to get started with them.

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Topics: Plasmid Technology, Plasmids 101

Choosing Your Perfect Empty Backbone

Posted by Lianna Swanson on Aug 19, 2014 11:39:33 AM

Vectors (or empty backbones) are frequently used in molecular biology to isolate, multiply, or express the insert they carry in the target cell. These vectors allow you to test the function of Your Gene Of Interest (YGOI) in a controlled environment under various conditions. The first thing you'll need to decide when running your experiment, is which vector will best suit your needs?

At Addgene, we have a vast collection of empty backbones that have been designed, tested, and published by academic scientists. To help you find the vector that fits your experiments, I've described below some of the most frequently requested vectors in our repository and will discuss some of the features you may want to consider as you make your choice.

The first and most important thing you need to know is your expression system or environment. The host organism will determine the type of vector that you will need. You will also have to make sure that your plasmid has been incorporated into the host organism, usually achieved with the proper selection marker or antibiotic resistance.

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Topics: Plasmid How To, Plasmid Technology, Plasmid Elements

Synthetic Biology & the Importance of Assembly Standards

Posted by Guest Blogger on Jul 8, 2014 11:06:00 AM

The field of synthetic biology has seen tremendous growth in recent years. At Addgene, synthetic biology deposits have grown exponentially, from just 2 plasmids in 2005 to 439 plasmids deposited last year. To shed some light on this growing field, we asked our friends at iGEM to share their expertise and discuss the importance of standards in the field.

The following post was contributed by Kim de Mora, iGEM Fellow.

What is Synthetic Biology?

“What I cannot create, I do not understand.” – Richard Feynman

This Feynman quote perfectly embodies the aims of synthetic biology in a single sentence. During the history of humanity, some of the most complex devices we have constructed are nuclear submarines, the space shuttle, the international space station and the Internet. But in all our existence, we have yet to design, engineer and build a cell from the ground up. A single bacterial cell is orders of magnitude more complex that the aforementioned feats of mechanical, aeronautical, electrical and computer engineering. These devices could be built because the underlying physical model of how the world works is understood by scientists and applied by engineers to practical ends.

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Topics: Plasmid Technology, Synthetic Biology

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