Product Class: Kit

NEBridge® Golden Gate Assembly Kit (BsmBI-v2)

Complement this kit with NEBridge Golden Gate Assembly Kit (BsaI-HFv2) (NEB #E1601)


Catalog #E1602

Product Introduction

Learn about ligase fidelity and push the limits of your Golden Gate Assembly Reactions (50+ fragments)

  • Uses BsmBI-v2, which is optimized for Golden Gate Assembly
  • Use for seamless cloning – no scar remains following assembly
  • Ideal for ordered assembly of multiple fragments (2-50+) in a single reaction
  • Can also be used for cloning of single inserts and library preparations
  • Efficient with regions with high GC content and areas of repeats
  • Compatible with a broad range of fragment sizes (< 100 bp to > 15 kb)
  • Includes destination plasmid compatible with a variety of Type IIS restriction enzymes commonly used in Golden Gate Assembly
  • Free tool available at GoldenGate.neb.com
  • Try our free Ligase Fidelity Tools ( for the design of high-fidelity Golden Gate Assemblies)

Product Information

Description

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.

To learn more about the Golden Gate Assembly workflow, watch this video tutorial. 

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.
This product is related to the following categories:
DNA Assembly, Cloning and Mutagenesis Kits Products
This product can be used in the following applications:
DNA Assembly and Cloning,
High-throughput cloning and automation solutions,
NEBridge® Golden Gate Assembly

Properties & Usage

Materials Required but not Supplied

  • User-defined inserts
  • Competent cells
  • Other materials for transformation

Features

  • Seamless cloning – no scar remains following assembly
  • Ordered assembly of multiple fragments in a single reaction
  • Can also be used for cloning of single inserts
  • Efficient with regions with high GC content and areas of repeats
  • Compatible with a broad range of fragment sizes (< 100 bp to > 15 kb)
  • Free tool available at GoldenGate.neb.com
  • 2 year shelf life

References

  1. Engler, C. et al. (2008). PLoS ONE. 3, e3647.
  2. Engler, C. et al. (2009). PLoS ONE. 4, e5553.
  3. Lee, J.H. et al. (1996). Genetic Analysis: Biomolecular Engineering. 13, 139–145.
  4. Padgett, K.A. and Sorge, J.A. (1996). Gene. 168, 31–35.
  5. Weber, E. et al. (2011). PLoS ONE. 6 , e19722.
  6. Christian, M. et al. (2010). Genetics. 186, 757–761.
  7. Pullman, P. et al. (2019). Nature. 9, 10932.
  8. Potapov, V. et al. (2018). ACS Synth. Biol. 7, 2665–2674.

Protocols, Manuals & Usage

Protocols

  1. Golden Gate Assembly Protocol for using NEBridge® Golden Gate Assembly Kit (BsmBI-v2) (NEB #E1602)
  2. Transformation Protocol for NEBridge® Golden Gate Assembly Kit (BsmBI-v2) (NEB #E1602)
  3. Recommended Screening Protocols for NEBridge® Golden Gate Assembly Kit (BsmBI-v2) (NEB #E1602)
  4. 35-Fragment Golden Gate Assembly using BsmBI-v2 (NEB #E1602)

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

Usage & Guidelines

Application Notes

Tools & Resources

Selection Charts

Web Tools

FAQs & Troubleshooting

FAQs

  1. What is the mechanism for Golden Gate Assembly?
  2. Which kit from NEB should I use for Golden Gate Assembly—the BsmBI-v2 kit (NEB #E1602) or the original BsaI-HFv2 kit (NEB #E1601)?
  3. What if there are internal BsaI and BsmBI sites in my insert sequences?
  4. The Type IIS restriction enzyme used in this kit is BsmBI-v2. How does it compare to the original BsmBI or Esp3I?
  5. How does the performance of this kit compare to “home brews” built with individual Type IIS restriction enzymes and T4 DNA Ligase components?
  6. What affects the efficiency of Golden Gate Assembly?
  7. Why do many of the published Golden Gate Assembly articles feature precloned inserts as opposed to inserts generated by PCR?
  8. Using purified amplicons directly without precloning seems much easier, but is the assembly efficiency decreased?
  9. Can PCR amplicons be used directly in assembly reactions without purification?
  10. Why is Golden Gate also used for single insert cloning?
  11. Why do assembly reactions end with a 5 minute, 60°C incubation step?
  12. How can I minimize PCR-generated errors in my amplicon inserts?
  13. What is an appropriate negative control for Golden Gate Assembly?
  14. How many cycles are optimal?
  15. Can I use other competent E. coli strains than NEB 10-beta? Can I use subcloning efficiency cells?
  16. How can I access pGGAselect as a GenBank or FASTA file?

Tech Tips

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:

  • Ligase Fidelity Viewer-visualize overhang ligation preferences
  • GetSet™ – predict high-fidelity junction sets
  • SplitSet™ – split DNA sequence for scarless high-fidelity assembly

All tools are available at neb.com/research/NEBeta-tools

 

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.

 

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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