Golden Gate cloning is a single tube method for rapidly and efficiently creating multi-insert plasmid constructs (Englers et al 2008). It is the primary method the Nonet lab uses to create integration plasmids for RMCE and other similar genome manipulation techniques. Here I provide a list of reagents that we have created to facilitate use of this methodology. I hope in the near future to make these plasmids available as a kit (in 96 well format as DNA). For the moment individual or groups of plasmids can be requested by emailing M. Nonet.
This framework for Golden Gate cloning involves using a combination of DNAs (usually plasmids or PCR fragments) that contain compatible SapI restriction sites to assemble a final product that lacks SapI sites. This is made possible because SapI cuts outside of its recognition sequence and leaves a 3 bp overhang. Since the final product does not contain any SapI sites, the ligation and digestion can be repeated many times by alternating cycles of 37°C to favor digestion and 16°C to favor ligation. This makes the protocol exceedingly efficient and reliable (Figure 1).
The specific implementation we have adopted is a derivative of, highly dependent on, and mostly compatible with the tools and methods developed by the work of Dickinson et al. (2015), Schwartz and Jorgensen (2016) and Dickinson et al (2018). Details about the similarities and differences between our implementation and those listed above can be found in Knoebel et al. (2023).
The approach can be used to create a variety of different multi-insert integration plasmids such as ones encoding promoter::FP, promoter::gene::FP and promoter::FP::gene fusions (Figure 2). The Nonet lab has developed clone libraries of promoters, fluorescent proteins, indicators, tags , linkers and 3′ UTRs, which can be used in combination to simplify creating new clones using GG assembly.
This approach can also be used to create targeting vectors for CRISPR mediated genome modification. In these cases, we use the TGG GCG and ACG TGA slots for the arms of the targeting vector and use floxed HygR cassettes in the NT slot as one of the inserts. A few complete assemblies are also available. We assemble these targeting plasmids in a vector containing an FRT site and inject into a strain expressing germline FLP (Nonet, 2023) to eliminate the potential formation of arrays and potentially increase availability of templates for repair.
In addition to the library, we have also developed vectors to simplify creation of new insert clones using BsaI mediated Golden Gate cloning. The lab generally prefers to expend the effort to create a KanR insert clone and sequence the resulting clone rather than use a PCR product. If the sequence of insert clones has been confirmed, we find it unnecessary to sequence the final SapI GG product. A manual detailing the approach and protocols we use to create both insert clones and RMCE integration clones is also available.
Finally, to aid in designing clones we have created an Excel based GG vector assembly tool that permits one to select among all available plasmids for each of the different entry slots and output either a sequence or a Genbank annotated sequence which can be pasted into a word file, then opened using ApE (Davis and Jorgensen, 2022) or other DNA sequence analysis software. Two versions of the tool are available. GGAssemblyBuilder contains the vectors and entry clones described in Knoebel et al. (2023), and GGNAssemblyBuilder contains those clones plus the vast majority of other clones created by the Nonet lab. Requests for clones should be sent to MLN.
Vector clones
Insert clones
CRIPSR/cas9 targeting vector assembly clones
Excel Golden Gate Assembly Tools (downloads)
We strive to provide accurate information about the plasmids and methods we have developed. Please let us know if you find an error, find missing or errant links, find statements in this webpage erroneous or confusing, or have other suggestions that could improve this website.
References
Engler C, Kandzia R, Marillonnet S (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS One (3): e3647. PMID:18985154
Davis MW, Jorgensen EM (2022) ApE, A Plasmid Editor: A Freely Available DNA Manipulation and Visualization Program. Front Bioinform (2): 818619. PMID:36304290
Dickinson DJ, Slabodnick MM, Chen AH, Goldstein B (2018) SapTrap assembly of repair templates for Cas9-triggered homologous recombination with a self-excising cassette. microPublication Biology DOI 10.17912/W2KT0N
Knoebel, E., Dour, S., & Nonet, M. L. (2023). A toolkit for assembly of targeting clones for C. elegans transgenesis. microPublication Biology. 10.17912/micropub.biology.000966.
Nonet ML (2020) Efficient transgenesis in C. elegans using Flp recombinase-mediated cassette exchange. Genetics (215): 902-921. PMID:32513816
Nonet ML (2023) Rapid generation of C. elegans single-copy transgenes combining RMCE and drug selection. Genetics iyad072. PMID:37079426
Schwartz ML, Jorgensen EM (2016) SapTrap, a Toolkit for High-Throughput CRISPR/Cas9 Gene Modification in Caenorhabditis elegans. Genetics (202): 1277-1288. PMID:26837755
last updated 10-2-2023