The NEBridge Golden Gate Assembly Kit (BsmBI-v2) contains an optimized mix of BsmBI-v2 and T4 DNA Ligase. BsmBI-v2 has been engineered by NEB and outperforms BsmBI in Golden Gate Assemblies. Together these enzymes can direct the assembly of multiple inserts/modules and also single insert/library generation cloning with single insert(s) using the Golden Gate approach. The pGGAselect destination plasmid is also provided, which provides a backbone for your assembly. This versatile destination construct has flanking recognition sites in the correct orientation for BsmBI-directed assemblies, and also BsaI- and BbsI-directed assemblies, enabling the destination plasmid to conveniently be used with all three of the most commonly used Type IIS restriction enzymes used for Golden Gate Assembly. It features convenient restriction enzyme sites for subcloning, and has T7/SP6 promoter sequences to enable in vitro transcription.
The efficient and seamless assembly of DNA fragments, commonly referred to as Golden Gate Assembly (1,2), had its origins in 1996, when for the first time it was shown that multiple inserts could be assembled into a vector backbone using only the sequential (3) or simultaneous (4) activities of a single Type IIS restriction enzyme and T4 DNA Ligase.
Type IIS restriction enzymes bind to their recognition sites but cut the DNA downstream from that site at a positional, not sequence- specific, cut site. Thus, a single Type IIS restriction enzyme can be used to generate DNA fragments with unique overhangs. As an example, BsmBI has a recognition site of CGTCTC(N1/N5), where the CGTCTC represents the recognition/binding site, and the N1/N5 indicates the cut site is one base downstream on the top strand, and five bases downstream on the bottom strand. Assembly of digested fragments proceeds through annealing of complementary four base overhangs on adjacent fragments. The digested fragments and the final assembly no longer contain Type IIS restriction enzyme recognition sites, so no further cutting is possible. The assembly product accumulates with time.
While particularly useful for multi-fragment assemblies such as Transcription Activator Like Effectors (TALEs)(5) and TALEs fused to a FokI nuclease catalytic domain (TALENs)(6), the Golden Gate method can also be used for cloning of single inserts and inserts from diverse populations that enable library creation, and multi-site mutagenesis involved in directed evolution (7).
Please note that while general descriptions regarding Golden Gate Assembly use the BsmBI nomenclature, this kit and protocols feature the specific engineered form optimized for Golden Gate Assembly, BsmBI-v2.
Figure 1. Overview: Assembly Protocol of Golden Gate Assembly using BsmBI-v2
Figure 2. Golden Gate Workflow
In its simplest form, Golden Gate Assembly requires a Type IIS recognition site, in this case, (CGTCTC), added to both ends of a dsDNA fragment. After digestion, these sites are left behind, with each fragment bearing the designed 4-base overhangs that direct the assembly.
Figure 3. High Efficiency and High Fidelity Golden Gate Assembly with BsmBI-v2
Twenty-four fragment assemblies of a LacI/LacZ cassette were performed using the recommended cycling protocol for 11-20+ fragments. While 30 cycles is sufficient to achieve complex assemblies, the stability of the BsmBI-v2 and T4 DNA Ligase allows continued assembly through 65 cycles with a low background. (a) Efficiency of assembly and (b) accuracy of assembly (fidelity) vs cycle number.
Figure 4. Complex Assembly by BsmBI-v2 and T4 DNA Ligase
Twenty-four fragment assemblies of a LacI/LacZ cassette were performed using the recommended cycling protocol for 11-20+ fragments, with an extension to 65 cycles. Plating 1/10 of the outgrowth from transforming 2 µl of the 25 µl assembly reaction resulted in 1100 colonies with a 96% fidelity level, equivalent to 137,500 colonies per assembly reaction. This performance illustrates the stability of the enzyme mix that allows the option of cycling beyond the standard 30 cycle level if maximal assembly performance is desired.
Figure 5.
pGGAselect is a 2,220 bp cloning vector useful for Golden Gate Assembly. The plasmid contains two BsmBI restriction sites; digestion with BsmBI releases a 65 bp fragment and a 2,155 bp vector backbone fragment to receive your insert or assembly.
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Use of the NEB Golden Gate Assembly Tool (GoldenGate.neb.com) is strongly recommended; this tool will check insert sequences for internal Type IIS restriction enzyme sites and design primers to amplify your inserts for Golden Gate Assembly. The primers will feature 6 bases at the 5′ end flanking the recognition site, the recognition site itself, plus the 4-base overhangs that determine correct annealing and ligation of the inserts. All overhangs will automatically be designed as non-palindromic (to eliminate self-insert ligations), unique, and in the correct orientations to ensure correct assembly.
Research at NEB has led to an increased understanding of ligase fidelity, including the development of a comprehensive method for profiling end-joining ligation fidelity in order to predict which overhangs will result in greater accuracy (Potapov, V. et al. (2018) ACS Synth. Biol., 7, 2665–2674.). This ligase fidelity information can be used in conjunction with the NEB Golden Gate Assembly kits to achieve high efficiency and accurate complex assemblies. Please visit www.neb.com/GoldenGate for more information.
NEB has developed ligase fidelity tools to facilitate the design of high-fidelity Golden Gate Assemblies:
Standard Golden Gate protocol suggests using 30 cycles, alternating between restriction and cutting. BsaI-HFv2, BsmBI-v2 and T4 DNA Ligase are very stable, allowing cycling up to 60 cycles, with high efficiency and fidelity. Consider whether your workflow would be enhanced by adding more cycles.
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