I make extensive use of key publications to help people understand the tools that I use for building plasmids via Type IIS restriction endonuclease mediated assembly (Golden Gate cloning). For some time I have desired to make modifications to Figure 2 from Engler et al., ACS Synthetic Biology (2014) v3 839-843. While the original cartoon is a good starting point for explaining the ins and outs of Golden Gate, on more than one occasion I have had the person I am speaking with become completely confused about the 3′ end of the illustrated molecules. I finally have made modifications to the image, and I hope that these will help clarify that the convention used in the Golden Gate world, whereby the “motif” or “fusion” between two pieces of DNA refers ONLY to the top strand, while the actual sequence of the 5′ overhang that is produced by BsaI digestion at the 3′ end of the molecules is actually the reverse complement of that which is shown. Additionally, I tried to impart some sense of directionality to the Type IIS enzymes by inverting the labels for those recognition motifs which reside in the bottom strand of the DNA. Finally, since color printers tend to produce a bit darker result than what one views on a computer screen, I opted to exchange the black text for white in selected colored bars used for the Level 0 components. Hopefully this will be useful for your comprehension of Golden Gate cloning.
On November 20th, 2017 I began my new position as the Molecular Technologies Department Research Manager at the Wisconsin Crop Innovation Center. This remarkable facility, gifted from Monsanto to the University of Wisconsin – Madison at the beginning of 2017, is now the largest publically held plant transformation facility in the United States. I am very fortunate to be able to join the team in the early stages of development of what will hopefully grow to become the premier plant transformation center in the world. I hope that my contributions will help toward that effort, and I cannot wait to see what new discoveries we make. I am grateful to my previous post-doctoral mentors for providing me the opportunity to develop skills and resources that have allowed me to land this job, and look forward to continuing to work with each of them in my new capacity.
Very nice animation, describing CRISPR/Cas9, recently released on YouTube by Nature Methods.
In the process of designing improvements to some of the tools under development in the lab, as well as engineering a fix for the Modular Cloning kit Level 2 position 5 end linker debacle, I have been making use of the valuable information housed in the Registry of Standard Biological Parts. This remarkable resource provides a vast array of DNA sequences, many of which have been functionally validated, that can be readily adapted for your specific needs. If you are ever faced with a seemingly intractable problem, take a look in the iGEM inventory and see if your creativity isn’t sparked to devise a novel and creative solution or work-around.