All Posts

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)

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.

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.

This post was contributed by guest blogger Iris Lindberg, Professor at the University of Maryland School of Medicine.

In the Lindberg Lab we often make cell lines that overexpress genes of interest; more recently we have also been using Addgene CRISPR vectors to generate cell lines with knockouts of specific genes. Many years ago, people in the laboratory became frustrated with using glass cloning rings to isolate colonies of antibiotic-resistant cells; during the time required to grease, place and fill a dozen cloning rings, the remainder of the colonies on the plate dried out and died. The alternative to cloning rings, dilution cloning into 96-well plates, is extremely time- and resource-consumptive, since only wells with one cell can give rise to single clones, and thus many plates must be examined for single clones and then handled. Additionally, many cell lines, especially the endocrine cell lines we most commonly work with, require extra serum to survive at low densities - adding to the expense of dilution cloning.

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.