Easing the Protein Purification Process with pCri

Posted by Mary Gearing on Jun 19, 2015 11:08:00 AM

Protein purification can be one of the most stressful lab activities. Working with proteins requires a substantial amount of properly folded, relatively pure protein, but getting to this stage is often much easier said than done. As reviewed in our Plasmids 101 series, proteins are overexpressed from a plasmid construct, most often in special E. coli strains designed for protein expression. Cultures are then lysed and the protein of interest is purified using an affinity tag. Additional tags may be used to improve protein stability and solubility.

Determining the best way to express one’s protein of interest can save a lot of time later.

Read More >

Topics: Hot Plasmids, Plasmid Kits

22 Hot Plasmid Technologies from 2014

Posted by Joanne Kamens on Jan 6, 2015 12:21:53 PM

Updated Mini-transposon Vector for Bacterial Mutagenesis or Gene Targeting

Victor de Lorenzo's lab has engineered a modular mini-Tn5 vector that can be used to generate random mutagenesis libraries or to insert heterologous genes, reporters, or other markers into a target genome. They did this by selecting the important elements from existing transposon and vector systems and creating an all-synthetic vector that included only the elements needed for function.

The lab validated this vector, called pBAM1, by conducting random mutagenesis in the soil bacterium Pseudomonas putida and demonstrate that they can successfully create GFP fusion proteins with a variety of genes across the genome. Although this tool was published in 2011, it was only recently made available through Addgene and we want to highlight it for use in your research.

Check out Joanne's Reddit AMA

Read More >

Topics: Hot Plasmids, Lab Tips, Plasmid Kits

Introducing an All-in-One CRISPR/Cas9 Vector System for Multiplex Genome Engineering

Posted by Kendall Morgan on Nov 12, 2014 10:54:00 AM

A newly established all-in-one vector construction system for CRISPR/Cas9-mediated multiplex genome engineering is now available thanks to researchers at Japan’s Hiroshima University who described their new tool in Scientific Reports in June.

“The multiplexity is one of the most advantageous properties of CRISPR/Cas9 compared to ZFNs and TALENs,” said Tetsushi Sakuma of Hiroshima University. “However, there had been no systematically established way of making an all-in-one vector for multiplex genome engineering.”

Read More >

Topics: CRISPR, Plasmid Kits

3 Challenges in Plant Synthetic Biology

Posted by Guest Blogger on Jul 22, 2014 1:46:10 PM

This post was contributed by Nikolai Braun and Keira Havens, co-founders of Revolution Bioengineering. Read their previous blog post about how they started their company here.

The first transgenic plant was engineered over 30 years ago, but plant synthetic biology is still in its infancy. A long timeline from transformation to testing and a lack of well-characterized genetic tools make it challenging to engineer a specific function in these multicellular organisms. However, the rewards are great if you take the plunge – plants are the foundation of life on earth, and opportunities abound to build better fuels, feeds, foods, and fibers. And because working with plants can be challenging, there are a lot of unexplored areas in plant biotechnology that are ripe with opportunity. We’ve decided to jump into one of those unexplored areas with our color-changing flower, but to do that we’ve had to navigate the challenges involved in plant synthetic biology.

Read More >

Topics: Synthetic Biology, Plant Biology, Plasmid Kits

Designer PUF Proteins for Any RNA Target

Posted by Kendall Morgan on Jun 17, 2014 3:57:00 PM

With the meteoric rise of CRISPR technology, the ability to direct enzymes – from nucleases to transcription factors – to specific sequences of DNA has become commonplace. This ability has opened up a world of possibilities in the engineering of complex gene networks. A comparable system for targeting specific sequences of RNA is highly desirable for extending the complexity of genetic circuits, allowing for tighter spatio-temporal control of gene expression within a cell. Thanks to the work of Huimin Zhao and colleagues, we now have just the tool…designer PUF proteins!

A newly available PUF Assembly Kit makes it possible to devise RNA binding proteins to hit any target of interest. The new tool was developed and implemented by applying the Golden Gate cloning method to human proteins known as Pumilio/fem-3 mRNA binding factors (PUF). In a single step, researchers can now assemble designer PUF domains for RNA specificity engineering.

“The RNA binding domain is interesting because by changing certain amino acids you can change the specificity,” explained Zhanar Abil of the University of Illinois at Urbana-Champaign.

Read More >

Topics: Plasmid Technology, Synthetic Biology, Plasmid Kits

Addgene blog logo

Subscribe to Our Blog