Plant tissue culture is a laborious endeavor. Well optimized protocols have been established for the production of healthy transgenic plants. Success is dependent on many factors, not least of which is the medium in which the plant tissue is cultured. Remarkably, the gelling agent can alter the pH of the culture medium. In this YouTube video, from the manufacturer PhytoTechnology Laboratories, pH adjustment is made after the gelling agent has been added. Interestingly, PhytoTechnology Laboratories provided feedback, in the comments below the video, indicating that many gelling agents contain sodium hydroxide (which they contend is utilized for gel strength). We confirmed the necessity of this procedure by monitoring the pH of a batch of callus induction medium for Brachypodium distachyon. The pH of a solution of Linsmaier & Skoog salts and maltose in ultrapure water was 3.4; the addition of the appropriate amount of Phytagel resulted in an increase of the pH to 5.0! Given that the pH scale is logarithmic, this is a very large increase.
One of the recurring issues with comprehension by my students of Golden Gate cloning seems to revolve around the nature of DNA. It takes practice to think upside down and backwards, which is necessary because of the reverse complementary nature of double stranded DNA. The papers which describe Golden Gate cloning are great and really helpful references, but there is a figure in one paper in particular that could use a small edit to help with comprehension. In the paper “A Modular Cloning System for Standardized Assembly of Multigene Constructs” by Weber et al (PLoS One (2011) v.6 e 16765 doi:10.1371/journal.pone.0016765) Figure 5 displays the top strand nucleotides to describe the fusion sites for the reverse orientation Level One acceptor plasmids. While these residues are absolutely correct, my students become confused since this labeling is inconsistent with that utilized for the forward version of the plasmid, as well as the labeling used to describe the 5′ terminus of the Level Zero promoter, and the 3′ terminus of the Level Zero terminator. In an effort to help my students and other end users better understand the reverse acceptor molecules I have adapted a portion of the figure, by displaying the reverse complement strand (with the text shown in an upside down orientation in the middle portion of the figure below). Additionally, I have provided the double stranded DNA sequence found in these regions to better illustrate the sites. It is my hope that by utilizing labeling more consistent with that utilized for the forward plasmid, as well as the companion Level Zero plasmids providing parts for incorporation into the acceptors, that comprehension is improved for mastery of this great system.
I am tasked with management of our large and rapidly growing collection of plasmids used for construction of vectors for manipulation of gene expression in both dicot and monocot plants. We are fully committed to Golden Gate cloning (which utilizes Type II-S restriction endonucleases (eg. BsaI, BpiI or BsmBI) for cutting DNA, in contrast to Type II-P enzymes (eg. BamHI, EcoRI, or HindIII) used for more traditional molecular biology). One of the keys to success in Golden Gate cloning is taking advantage of differences in antibiotic resistance genes between plasmids of different levels. In an effort to help end users quickly identify the antibiotic resistance background of any of the plasmids in our collection, I instituted a simple color scheme for the glycerol stock tubes. This simple strategy can help to eliminate confusion and ensure successful construction of transcriptional units and binary vectors.
Prior to starting my current position I was reliant on Type IIP (palindromic) restriction endonucleases for all of my cloning experiments for construction of plasmids to modify gene expression in plants. Type IIS (staggered) enzymes, in conjunction with excellent pricing for synthetic molecules, have been a welcome change to my toolbox, liberating me from the constraining limitations imposed by reliance on natural or PCR incorporated TypeIIP motifs for cobbling together DNA fragments. The MoClo and MoClo Plant Parts kits (available from Addgene) have truly changed how I approach any given project. These 5 papers will help to get started in Golden Gate cloning:
- Engler, C., Kandzia, R., Marillonnet, S., Eldik, G. Van and Botterman, J. (2008) A One Pot, One Step, Precision Cloning Method with High Throughput Capability H. A. El-Shemy, ed. PLoS One, 3, e3647.
- Engler, C., Youles, M., Gruetzner, R., Ehnert, T.-M., Werner, S., Jones, J.D.G., Patron, N.J. and Marillonnet, S. (2014) A golden gate modular cloning toolbox for plants. ACS Synth. Biol., 3, 839–43.
- Patron, N.J., Orzaez, D., Marillonnet, S., et al. (2015) Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytol., 208, 13–9.
- Weber, E., Engler, C., Gruetzner, R., Werner, S. and Marillonnet, S. (2011) A Modular Cloning System for Standardized Assembly of Multigene Constructs J. Peccoud, ed. PLoS One, 6, e16765.
- Werner, S., Engler, C., Weber, E., Gruetzner, R. and Marillonnet, S. (2012) Fast track assembly of multigene constructs using Golden Gate cloning and the MoClo system. PLoS One, 3, 38–43.