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Harnessing Bacterial Toxins for Allelic Exchange

Posted by Guest Blogger on Aug 15, 2019 8:30:02 AM

This post was contributed by Jacob Lazarus, a postdoctoral researcher at Harvard.

There’s an astounding number of ways to create chromosomal mutations in bacteria, so many that it may be difficult to decide which path to take. A quick and easy way to introduce a mutation in the chromosome is to disrupt expression of a gene with an antibiotic resistance cassette. This leaves a “scar” in the chromosome, sometimes interfering with expression of surrounding genes. However, there are ways to create scarless mutations, ones that don’t leave any undesired scars in the chromosome.

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Topics: Plasmid Protocols and Tips, Other Plasmid Tools, Plasmids

Save Time with Transient Plant Leaf Transformations

Posted by Guest Blogger on Jul 25, 2019 8:03:52 AM

This post was contributed by Samuel Mortensen, a PhD candidate at Northeastern University.

Working with plants doesn’t always have to be a time-consuming process. While developing transgenic hairy root lines in tissue cultures takes half a year, and generating a transgenic plant can take even longer, a transient plant leaf transformation process could save the plant biologist some time… months, in fact.

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Topics: Plant Biology, Plasmid Protocols and Tips, Other

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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