In my capacity as the Research Manager of the Molecular Technologies Department within the Wisconsin Crop Innovation Center, part of the University of Wisconsin – Madison, I am engaged in several projects in which I utilize Golden Gate cloning. Since first adopting Golden Gate cloning during my post-doctoral work in the Ané lab at the UW, I have worked to develop new TypeIIS mediated cloning tools, to expand the applicability of the MoClo Plant Parts kit, for constructing plant molecular biology focused transcriptional units and binary plasmids. I have noticed during the past year that every time I search for information regarding TypeIIS enzymes, or Golden Gate cloning that that I find exactly the paper I am looking for in the literature, with publication dates are often only months or weeks before I have searched for them. I am very happy to be able to tap into such a vibrant and active area of research. The latest examples are a couple of outstanding papers recently published by the fine folks at New England Biolabs (pub 1) (pub 2). These manuscripts provide a treasure trove of empirical data, related to ligation efficiency and fidelity of 4 nucleotide overhanging sequences, that anyone engaged in non-traditional Golden Gate cloning design would do well to read and use in their future work.
After a long waiting period, the work completed during my post-doc with the USDA in California was finally published in 2018. Our unconventional approach to Agrobacterium-mediated plant transformation was met with skepticism and our manuscript was rejected by several journals. Finally, the sacrifice of nearly all my weekends during 2015 and 2016 was rewarded when our manuscript was accepted. The cherry on top was the selection of our work, including a cover image, as Featured Article in The Plant Journal (volume 95, issue 4, August 2018; https://doi.org/10.1111/tpj.13992).
The Molecular Technologies Department that I manage at the Wisconsin Crop Innovation Center, of the University of Wisconsin – Madison, has been fortunate to receive internal funding from the University of Wisconsin – Madison for instrumentation to help us to achieve our goals in helping UW researchers conduct their research. One of the devices that I have installed in the laboratory is an oKtoputre DNA extraction robot, manufactured by LGC (formerly Douglas Scientific). I made and posted a short video of the robot in operation. I recently utilized this well made robot (which I have named “Stephen”, in honor of Stephen Curry, since the robot does a little “shimmy” every time if ejects or picks up tips) to extract DNA from four 96 well plates containing soybean leaf samples from plants that are part of a CRISPR/Cas9 gene editing experiment. The entire procedure took ~ 2 hours, with another hour for cleaning. With the robot and DNA extraction protocol well established, we now offer genomic DNA extraction as one of our fee for service offerings. I hope that this unit will help many researchers to push forward with their projects.
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.