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Plasmids 101: Biotinylation

Posted by Alyssa Cecchetelli on Nov 15, 2018 8:50:12 AM

Biotin and its binding partner avidin are commonly used today in molecular biology for an array of different techniques and protocols. In this post we will discuss the natural role of biotin, biotinylation, the discovery of the biotin-avidin interaction and the uses of biotinylation in molecular biology!

Learn about in vivo biotinylation of bacterial fusion proteins

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Topics: Plasmids 101, Plasmids

Bacterial Toxin-antitoxin Systems as Molecular Biology Tools

Posted by Jennifer Tsang on Nov 1, 2018 8:35:58 AM

Members of the bacterial world produce an assortment toxins to claim territory or kill competing microorganisms, but did you know bacteria also produce substances toxic to themselves?

What are toxin-antitoxin systems?

These toxic substances are part of toxin-antitoxin systems that are widely present in bacteria. They consist of a toxin which can affect a variety of cellular processes and an antitoxin that suppresses the toxin’s activity. The key to these systems is that the toxin is stable while the antitoxin is unstable, meaning that the cells must continually produce antitoxin to avoid cell death.

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Topics: Plasmid Elements, Plasmids

Tips for arabidopsis transformation

Posted by Guest Blogger on Oct 25, 2018 9:23:48 AM

This post was contributed by Laura Lee, a graduate student at Stanford University.

Arabidopsis is a fantastic model organism for many reasons, not the least of which is ease of transformation. There are many motivations to generate transgenic Arabidopsis, from studying transcriptional and translational dynamics of genes and proteins in living plants, to complementing mutant phenotypes. Arabidopsis is amenable to the floral drip or dip transformation method. The general steps for this method include:

  • Cloning and transforming a plasmid into the bacterium Agrobacterium tumeficans - a plant pathogenic species that stably integrates transfer DNA (tDNA) into the genomes of the plants it attacks
  • Growing the transformed agrobacterium culture
  • Dipping your plant’s flowers in the agrobacterium culture to allow for tDNA insertions into the plant’s germline
  • Selecting for seeds that have the tDNA insertions (usually via seed growth on antibiotic-containing media)
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Topics: Plant Biology, Plasmid Protocols and Tips, Plasmids

Simplify Cloning with in vivo Assembly

Posted by Guest Blogger on Oct 18, 2018 8:37:05 AM

This post was contributed by Jake Watson and Javier García-Nafría from the MRC Laboratory of Molecular Biology.

Plasmid cloning is an essential part of any molecular biology project, yet very often, it is also a bottleneck in the experimental process. The majority of current cloning techniques involve the assembly of a circular plasmid in vitro, before transforming it into E. coli for propagation. However, while not widely known, plasmid assembly can be achieved in vivo using a bacterial recombination pathway that is present even in common lab cloning strains.

This intrinsic bacterial recombination pathway, referred to as recA-independent recombination, joins together pieces of linear DNA through short homologous sequences at their termini, and likely functions as a bacterial DNA repair mechanism. The pathway is ubiquitous, with successful recombination reported in all laboratory E. coli strains tested so far.

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Topics: Plasmid Cloning, Plasmid Protocols and Tips, Plasmids

Golden Gate Assembly upgrades: More fragments, faster assembly, and higher fidelity

Posted by Guest Blogger on Oct 11, 2018 8:30:35 AM

This post was contributed by guest bloggers Becky Kucera, M.Sc. and Eric Cantor, Ph.D. from New England Biolabs.

Golden gate assembly limitations

Embraced by the synthetic biology community, Golden Gate Assembly is commonly used to assemble 2–10 DNA fragments in a single “one-pot” reaction to form complex, multi-insert modular assemblies that enable biosynthetic pathway engineering and optimization. However, current best practices for assemblies of more than 10 modules often rely on two-step hierarchical approaches using different Type IIS restriction enzyme specificities at each step. Factors such as enzyme efficiency, stability, and buffer compatibility have placed practical limits on single- or two-step assemblies.

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Topics: Plasmid Cloning, Plasmid Protocols and Tips, Plasmids

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