First described in the 1980s, protein tags are now one of the most useful items in a scientist’s toolbox. As we’ve covered in Plasmids 101, tags can help you determine localization of a protein of interest, purify it, or determine its expression level without the need for a custom antibody. There is one major caveat - a tag may interfere with protein localization and/or function, so each tagged protein must be tested carefully to ensure it retains the attributes of the native protein. Since this process takes a lot of time and energy, Martí Aldea and collaborators have created a set of “innocuous tags” (inntags) less likely to alter a protein’s properties.
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.
Exciting news! Addgene recently rolled out a new feature on our plasmid pages - links to articles citing this plasmid. Now you can learn how a plasmid has been used by multiple labs and see what experimental systems it has been validated in.
If a plasmid's Addgene ID # has been referenced in other publications, you'll find a link to the list of citing articles under the "Resource Information" heading in the right column of the plasmid page. Check out the purple arrow in the screenshot below to see what I mean.
Once you've clicked on the "# References" link under the "Resource Information" heading, you'll be directed to a page listing the articles that cite this plasmid. You can use the dropdown to increase the length of the list (purple oval in the screenshot below). You can also use the "Search Table" box at the upper right of the table to search and filter the list of citing articles. From the article list you can click on the PubMed link to find the article abstract and more.
Addgenie Eric Perkins attended the recent Keystone Meeting "Precision Genome Engineering and Synthetic Biology". His reflections on the program are here. This was a great opportunity for Addgene to present our own data on plasmid deposits and distirbution for these fast moving fields.
Addgene is a global nonprofit plasmid repository. Over 2,000 labs have deposited plasmids to Addgene and we distribute over 130,000 plasmids in 2014. Thus, we are in a unique position to observe and quantify how new technologies are being disseminated through the scientific community.
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.