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Tips for Screening with Yeast Two Hybrid Systems

Posted by Jessica Welch on Oct 22, 2015 10:30:00 AM

Two hybrid systems were developed in Saccharomyces cerevisiae in 1989 and are still used extensively to screen for molecular interactions in the cell, including protein-protein, protein-DNA and protein-RNA interactions.

The 1980s saw a flurry of discovery in the field of eukaryotic transcriptional activation and cell biology. During this period, proteins were successfully expressed as fusions that retain their individual activities (1). Researchers also discovered the modular format of some transcriptional activators: that the DNA binding domain (DBD) and transcriptional activation (TA) domains retain their individual activities when separated (2), and that DBD and TAs from different systems could be combined effectively (3).

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Topics: Plasmid Technology, Yeast

pSiM24: Simplifying Plant Genetic Engineering

Posted by Mary Gearing on Sep 29, 2015 10:30:00 AM

As previous blogs have noted, plants are an important foundation for life on Earth. Selective breeding methods have shaped the plants that we grow and eat, and genetic engineering will continue to improve plant nutrition, yield, and pest resistance. Much of plant genetic engineering revolves around Agrobacterium tumifaciens. Agrobacterium carries a “tumor-inducing” or Ti plasmid, which allows it to transfer genetic material into the host plant genome. Scientists have worked to optimize this system for gene transfer, studying the stability of modified Ti plasmids during plant infection, as well as plasmid yield during preparation in E. coli. Addgene depositor Indu Maiti has created a new and versatile binary Ti vector for both transient and stable gene expression applications in plants. This smaller, easily customizable vector functions in multiple species, including tobacco and Arabidopsis.

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Topics: Plasmid Technology, Plant Biology

Sleeping Beauty Awakens for Genome Engineering

Posted by Emma Markham on Jun 30, 2015 10:00:00 AM

Transposons are sequences of DNA that can move around in a genome. In a laboratory setting, transposons can be used to both introduce genes into an organism’s genome (see figure) and to disrupt endogenous genes at the site of insertion. In both of these cases, transposons combine the advantages of viruses and naked DNA while eliminating some of the drawbacks. Specifically, viruses are able to infect and replicate in host cells, but they are susceptible to cells’ defense mechanisms. The use of non-viral vectors, like transposons, avoids many, though not all, of these defenses. For some applications of genome engineering - such as certain forms of gene therapy - avoiding the use of viruses is also important for social and regulatory reasons.

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Topics: Plasmid Technology, Genome Engineering

Back to Bacteria: CRISPR gRNA Multiplexing Using tRNAs

Posted by Mary Gearing on Jun 2, 2015 2:06:00 PM

In the short time since its development, CRISPR/Cas9 genome editing has been used to study the effect of gene knockout in vivo and in vitro, as well as to insert targeted mutations through homologous recombination. To further increase the utility of CRISPR/Cas9, it will be necessary to improve its multiplexing capacity. Multiplexing is key due to the natural redundancy of biological pathways;  to observe a phenotype, the modification of multiple genes is often necessary.

Guide RNAs (gRNAs) are commonly packaged in 400-500 bp cassettes containing the RNA pol III promoter, gRNA and pol III terminator. These relatively large cassettes (considering the gRNA itself is ~100 bases) limit the number of gRNAs that can be packaged together in a single vector. In addition, the pol III promoter is relatively weak, and low expression of gRNAs from these constructs could lower genome editing efficiency.

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Topics: Plasmid Technology, Genome Engineering, CRISPR

Transgenic Organisms, Cas9 Gene Drives, and Appropriate Safeguards

Posted by Guest Blogger on May 22, 2015 12:58:12 PM

This post was contributed by Kevin Esvelt, a Wyss Technology Development Fellow at the Wyss Institute and Harvard Medical School.

Scientists making transgenic organisms with Cas9 should be aware of the potential hazards of creating “gene drives” capable of spreading through wild populations. Whereas most genomic changes impose a fitness cost and are eliminated by natural selection, gene drives distort inheritance in their favor and consequently can spread even when costly.

If even a single organism carrying a synthetic gene drive were to escape the laboratory, the drive could eventually spread through the entire wild population with unpredictable ecological effects. Because the consequences of such a mistake would necessarily extend far beyond the laboratory and seriously damage public trust in scientists, experiments involving potential gene drives should be conducted with extreme caution.

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Topics: Plasmid Technology, Lab Tips, CRISPR, CRISPR 101

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