Golden Gate Assembly

Background & Design:
Golden Gate Assembly (GGA) was first described in Engler C, Kandzia R, Marillonnet S (2008) and Engler C, Gruetzner R, Kandzia R, Marillonnet S (2009) as an efficient way to quickly assemble multiple DNA sequences, or parts, into a single plasmid. This molecular cloning method allows a researcher to simultaneously and directionally assemble multiple DNA fragments into a single piece using Type IIs restriction enzymes and T4/T7 DNA ligase. This assembly is performed in vitro. The most commonly used Type IIs enzymes are BsaI, BsmBI and BbsI. Unlike standard Type II restriction enzymes like EcoRI and BamHI, these enzymes cut DNA outside of their recognition sites and therefore can create customized overhangs. Since 256 potential overhang sequences are possible, multiple fragments of DNA can be assembled by using combinations of overhang sequences.

Golden Gate Assembly Links

General Golden Gate Assembly Protocols

• Golden Gate Assembly General Golden Gate assembly outside of the kit framework
• Tips, Tricks, and Troubleshooting Running into problems? Check out these potential solutions!

Bee Toolkit Golden Gate Assembly Scheme- Start to Finish

• Design a new part Design a new part with correct part-type overhangs.
• Assemble a new part plasmid Assemble a new part plasmid.
• Build a first-stage plasmid (Transcriptional Unit, BsaI assembly) Build a complete plasmid with a single BsaI reaction.
• Build a second-stage plasmid Assemble multiple first-stage transcriptional unit plasmids.

Useful Tools

• Bee Toolkit Primer Design: https://spleonard1.shinyapps.io/paRting/
• Reaction Calculation Spreadsheet: GGA_Kit_Reaction_Calculation_Spreadsheet.xlsx
• NEBeta Tools for the design of high-fidelity Golden Gate Assemblies: https://www.neb.com/research/nebeta-tools

• iGEM Alternate Part Plasmid Assembly

References

1. Lee ME, DeLoache WC, Cervantes B, Dueber JE. (2015) A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth. Biol. 4:975–986. Link
2. Engler C, Kandzia R, Marillonnet S. (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS ONE 3:e3647. Link
3. "Golden Gate Assembly". New England Biolabs. Retrieved 8 June 2015. Link
4. Iverson et al. (2016) CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology. ACS Syn Bio 5 (1), pp 99–103. http://pubs.acs.org/doi/abs/10.1021/acssynbio.5b00124

Contributors

• Peng Geng
• Sean Leonard
• Kate Elston
• Dennis Mishler
• Jeffrey Barrick

The excel worksheet for calculating Golden Gate Assembly Reactions can be found in the attachment below.

Golden Gate Assembly

Background & Design:
Golden Gate Assembly (GGA) was first described in Engler C, Kandzia R, Marillonnet S (2008) and Engler C, Gruetzner R, Kandzia R, Marillonnet S (2009) as an efficient way to quickly assemble multiple DNA sequences, or parts, into a single plasmid. This molecular cloning method allows a researcher to simultaneously and directionally assemble multiple DNA fragments into a single piece using Type IIs restriction enzymes and T4/T7 DNA ligase. This assembly is performed in vitro. The most commonly used Type IIs enzymes are BsaI, BsmBI and BbsI. Unlike standard Type II restriction enzymes like EcoRI and BamHI, these enzymes cut DNA outside of their recognition sites and therefore can create customized overhangs. Since 256 potential overhang sequences are possible, multiple fragments of DNA can be assembled by using combinations of overhang sequences.

• Design a new part Design a new part with correct part-type overhangs.
• Assemble a new part plasmid Assemble a new part plasmid.
• Build a first-stage plasmid (Transcriptional Unit, BsaI assembly) Build a complete plasmid with a single BsaI reaction.
• Build a second-stage plasmid Assemble multiple first-stage transcriptional unit plasmids.

• Bee Toolkit Primer Design: https://spleonard1.shinyapps.io/paRting/

• NEBeta Tools for the design of high-fidelity Golden Gate Assemblies: https://www.neb.com/research/nebeta-tools

iGEM-Specific Protocols

• iGEM Alternate Part Plasmid Assembly

References

1. Lee ME, DeLoache WC, Cervantes B, Dueber JE. (2015) A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth. Biol. 4:975–986. Link
2. Engler C, Kandzia R, Marillonnet S. (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS ONE 3:e3647. Link
3. "Golden Gate Assembly". New England Biolabs. Retrieved 8 June 2015. Link
4. Iverson et al. (2016) CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology. ACS Syn Bio 5 (1), pp 99–103. http://pubs.acs.org/doi/abs/10.1021/acssynbio.5b00124

Contributors

• Peng Geng
• Sean Leonard
• Kate Elston
• Dennis Mishler
• Jeffrey Barrick

The excel worksheet for calculating Golden Gate Assembly Reactions can be found in the attachment below.

Golden Gate Assembly

Background & Design:
Golden Gate Assembly (GGA) was first described in Engler C, Kandzia R, Marillonnet S (2008) and Engler C, Gruetzner R, Kandzia R, Marillonnet S (2009) as an efficient way to quickly assemble multiple DNA sequences, or parts, into a single plasmid. This molecular cloning method allows a researcher to simultaneously and directionally assemble multiple DNA fragments into a single piece using Type IIs restriction enzymes and T4/T7 DNA ligase. This assembly is performed in vitro. The most commonly used Type IIs enzymes are BsaI, BsmBI and BbsI. Unlike standard Type II restriction enzymes like EcoRI and BamHI, these enzymes cut DNA outside of their recognition sites and therefore can create customized overhangs. Since 256 potential overhang sequences are possible, multiple fragments of DNA can be assembled by using combinations of overhang sequences.

Golden Gate Assembly Schematic:

Golden Gate Assembly Links

Golden Gate Assembly - Start to Finish

• Design a new part Design a new part with correct part-type overhangs.
• Assemble a new part plasmid Assemble a new part plasmid.
• Build a first-stage plasmid (Transcriptional Unit, BsaI assembly) Build a complete plasmid with a single BsaI reaction.
• Build a second-stage plasmid Assemble multiple first-stage transcriptional unit plasmids.
• Tips, Tricks, and Troubleshooting Running into problems? Check out these potential solutions!

Useful Tools

• Bee Toolkit Primer Design: https://spleonard1.shinyapps.io/paRting/
• Reaction Calculation Spreadsheet: BroadHostRange_Reaction_Calculator.xlsx
• NEBeta Tools for the design of high-fidelity Golden Gate Assemblies: https://www.neb.com/research/nebeta-tools

• iGEM Alternate Part Plasmid Assembly

References

1. Lee ME, DeLoache WC, Cervantes B, Dueber JE. (2015) A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth. Biol. 4:975–986. Link
2. Engler C, Kandzia R, Marillonnet S. (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS ONE 3:e3647. Link
3. "Golden Gate Assembly". New England Biolabs. Retrieved 8 June 2015. Link
4. Iverson et al. (2016) CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology. ACS Syn Bio 5 (1), pp 99–103. http://pubs.acs.org/doi/abs/10.1021/acssynbio.5b00124

Contributors

• Peng Geng
• Sean Leonard
• Kate Elston
• Dennis Mishler
• Jeffrey Barrick

The excel worksheet for calculating Golden Gate Assembly Reactions can be found in the attachment below.

Golden Gate Assembly

Background & Design:
Golden Gate Assembly (GGA) was first described in Engler C, Kandzia R, Marillonnet S (2008) and Engler C, Gruetzner R, Kandzia R, Marillonnet S (2009) as an efficient way to quickly assemble multiple DNA sequences, or parts, into a single plasmid. This molecular cloning method allows a researcher to simultaneously and directionally assemble multiple DNA fragments into a single piece using Type IIs restriction enzymes and T4/T7 DNA ligase. This assembly is performed in vitro. The most commonly used Type IIs enzymes are BsaI, BsmBI and BbsI. Unlike standard Type II restriction enzymes like EcoRI and BamHI, these enzymes cut DNA outside of their recognition sites and therefore can create customized overhangs. Since 256 potential overhang sequences are possible, multiple fragments of DNA can be assembled by using combinations of overhang sequences.

Golden Gate Assembly Schematic:

Golden Gate Assembly Links

Golden Gate Assembly - Start to Finish

• Design a new part Design a new part with correct part-type overhangs.
• Assemble a new part plasmid Assemble a new part plasmid.
• Build a first-stage plasmid (Transcriptional Unit, BsaI assembly) Build a complete plasmid with a single BsaI reaction.
• Build a second-stage plasmid Assemble multiple first-stage transcriptional unit plasmids.
• Tips, Tricks, and Troubleshooting Running into problems? Check out these potential solutions!

• Broad Host range Toolkit (BTK) - Derived from Leonard, Barrick 2018

iGEM-Specific Protocols

• iGEM Alternate Part Plasmid Assembly

References

1. Lee ME, DeLoache WC, Cervantes B, Dueber JE. (2015) A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth. Biol. 4:975–986. Link
2. Engler C, Kandzia R, Marillonnet S. (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS ONE 3:e3647. Link
3. "Golden Gate Assembly". New England Biolabs. Retrieved 8 June 2015. Link
4. Iverson et al. (2016) CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology. ACS Syn Bio 5 (1), pp 99–103. http://pubs.acs.org/doi/abs/10.1021/acssynbio.5b00124

Contributors

• Peng Geng
• Sean Leonard
• Kate Elston
• Dennis Mishler
• Jeffrey Barrick

The excel worksheet for calculating Golden Gate Assembly Reactions can be found in the attachment below.

Golden Gate Assembly

Golden Gate Assembly Links

Golden Gate Assembly - Start to Finish

• Design a new part Design a new part with correct part-type overhangs.
• Assemble a new part plasmid Assemble a new part plasmid.
• Build a first-stage plasmid (Transcriptional Unit, BsaI assembly) Build a complete plasmid with a single BsaI reaction.
• Build a second-stage plasmid Assemble multiple first-stage transcriptional unit plasmids.
• Tips, Tricks, and Troubleshooting Running into problems? Check out these potential solutions!

Kit-Specific Pages

• Broad Host range Toolkit (BTK) - Derived from Leonard, Barrick 2018

iGEM-Specific Protocols

• iGEM Alternate Part Plasmid Assembly

References

1. Lee ME, DeLoache WC, Cervantes B, Dueber JE. (2015) A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth. Biol. 4:975–986. Link
2. Engler C, Kandzia R, Marillonnet S. (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS ONE 3:e3647. Link
3. "Golden Gate Assembly". New England Biolabs. Retrieved 8 June 2015. Link
4. Iverson et al. (2016) CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology. ACS Syn Bio 5 (1), pp 99–103. http://pubs.acs.org/doi/abs/10.1021/acssynbio.5b00124

Contributors

• Peng Geng
• Sean Leonard
• Kate Elston
• Dennis Mishler
• Jeffrey Barrick

The excel worksheet for calculating Golden Gate Assembly Reactions can be found in the attachment below.

Golden Gate Assembly

Background & Design:
Golden Gate Assembly (GGA) was first described in Engler C, Kandzia R, Marillonnet S (2008) and Engler C, Gruetzner R, Kandzia R, Marillonnet S (2009) as an efficient way to quickly assembly multiple DNA sequences, or parts, into a single plasmid. This molecular cloning method allows a researcher to simultaneously and directionally assemble multiple DNA fragments into a single piece using Type IIs restriction enzymes and T4/T7 DNA ligase. This assembly is performed in vitro. The most commonly used Type IIs enzymes are BsaI, BsmBI and BbsI. Unlike standard Type II restriction enzymes like EcoRI and BamHI, these enzymes cut DNA outside of their recognition sites and therefore can create non-palindromic overhangs. Since 256 potential overhang sequences are possible, multiple fragments of DNA can be assembled by using combinations of overhang sequences.

Golden Gate Assembly Schematic:

Golden Gate Assembly Links

Golden Gate Assembly - Start to Finish

• Design a new part Design a new part with correct part-type overhangs.
• Assemble a new part plasmid Assemble a new part plasmid.
• Build a first-stage plasmid (Transcriptional Unit, BsaI assembly) Build a complete plasmid with a single BsaI reaction.
• Build a second-stage plasmid Assemble multiple first-stage transcriptional unit plasmids.
• Tips, Tricks, and Troubleshooting Running into problems? Check out these potential solutions!

Kit-Specific Pages

• Broad Host range Toolkit (BTK) - Derived from Leonard, Barrick 2018

iGEM-Specific Protocols

• iGEM Alternate Part Plasmid Assembly

References

1. Lee ME, DeLoache WC, Cervantes B, Dueber JE. (2015) A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth. Biol. 4:975–986. Link
2. Engler C, Kandzia R, Marillonnet S. (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS ONE 3:e3647. Link
3. "Golden Gate Assembly". New England Biolabs. Retrieved 8 June 2015. Link
4. Iverson et al. (2016) CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology. ACS Syn Bio 5 (1), pp 99–103. http://pubs.acs.org/doi/abs/10.1021/acssynbio.5b00124

Contributors

• Peng Geng
• Sean Leonard
• Kate Elston
• Dennis Mishler
• Jeffrey Barrick

The excel worksheet for calculating Golden Gate Assembly Reactions can be found in the attachment below.

Golden Gate Assembly

Background & Design:
Golden Gate Assembly (GGA) was first described in Engler C, Kandzia R, Marillonnet S (2008) and Engler C, Gruetzner R, Kandzia R, Marillonnet S (2009) as an efficient way to quickly assembly multiple DNA sequences, or parts, into a single plasmid. This molecular cloning method allows a researcher to simultaneously and directionally assemble multiple DNA fragments into a single piece using Type IIs restriction enzymes and T4/T7 DNA ligase. This assembly is performed in vitro. The most commonly used Type IIs enzymes are BsaI, BsmBI and BbsI. Unlike standard Type II restriction enzymes like EcoRI and BamHI, these enzymes cut DNA outside of their recognition sites and therefore can create non-palindromic overhangs. Since 256 potential overhang sequences are possible, multiple fragments of DNA can be assembled by using combinations of overhang sequences.

Golden Gate Assembly Schematic:

Golden Gate Assembly Links

Golden Gate Assembly - Start to Finish

• Design a new part Design a new part with correct part-type overhangs.
• Assemble a new part plasmid Assemble a new part plasmid.
• Build a first-stage plasmid (Transcriptional Unit, BsaI assembly) Build a complete plasmid with a single BsaI reaction.
• Build a second-stage plasmid Assemble multiple first-stage transcriptional unit plasmids.
• Tips, Tricks, and Troubleshooting Running into problems? Check out these potential solutions!

Kit-Specific Pages

• Yeast Toolkit (YTK) - Derived from Lee, Dueber 2015 (old Golden Gate Assembly page!)
• Broad Host range Toolkit (BTK) - Derived from Leonard, Barrick 2018

iGEM-Specific Protocols

• iGEM Alternate Part Plasmid Assembly

References

1. Lee ME, DeLoache WC, Cervantes B, Dueber JE. (2015) A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth. Biol. 4:975–986. Link
2. Engler C, Kandzia R, Marillonnet S. (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS ONE 3:e3647. Link
3. "Golden Gate Assembly". New England Biolabs. Retrieved 8 June 2015. Link
4. Iverson et al. (2016) CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology. ACS Syn Bio 5 (1), pp 99–103. http://pubs.acs.org/doi/abs/10.1021/acssynbio.5b00124

Contributors

• Peng Geng
• Sean Leonard
• Kate Elston
• Dennis Mishler
• Jeffrey Barrick

Golden Gate Assembly

Background & Design:
Golden Gate Assembly (GGA) was first described in Engler C, Kandzia R, Marillonnet S (2008) and Engler C, Gruetzner R, Kandzia R, Marillonnet S (2009) as an efficient way to quickly assembly multiple DNA sequences, or parts, into a single plasmid. This molecular cloning method allows a researcher to simultaneously and directionally assemble multiple DNA fragments into a single piece using Type IIs restriction enzymes and T4/T7 DNA ligase. This assembly is performed in vitro. The most commonly used Type IIs enzymes are BsaI, BsmBI and BbsI. Unlike standard Type II restriction enzymes like EcoRI and BamHI, these enzymes cut DNA outside of their recognition sites and therefore can create non-palindromic overhangs. Since 256 potential overhang sequences are possible, multiple fragments of DNA can be assembled by using combinations of overhang sequences.

Golden Gate Assembly Schematic:

Golden Gate Assembly Links

Golden Gate Assembly - Start to Finish

• Design a new part Design a new part with correct part-type overhangs.
• Assemble a new part plasmid Assemble a new part plasmid.
• Build a first-stage plasmid (Transcriptional Unit, BsaI assembly) Build a complete plasmid with a single BsaI reaction.
• Build a second-stage plasmid Assemble multiple first-stage transcriptional unit plasmids.
• Tips, Tricks, and Troubleshooting Running into problems? Check out these potential solutions!

Kit-Specific Pages

• Yeast Toolkit (YTK) - Derived from Lee, Dueber 2015 (old Golden Gate Assembly page!)
• Broad Host range Toolkit (BTK) - Derived from Leonard, Barrick 2018

iGEM-Specific Protocols

• iGEM Alternate Part Plasmid Assembly

References

1. Lee ME, DeLoache WC, Cervantes B, Dueber JE. (2015) A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth. Biol. 4:975–986. Link
2. Engler C, Kandzia R, Marillonnet S. (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS ONE 3:e3647. Link
3. "Golden Gate Assembly". New England Biolabs. Retrieved 8 June 2015. Link
4. Iverson et al. (2016) CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology. ACS Syn Bio 5 (1), pp 99–103. http://pubs.acs.org/doi/abs/10.1021/acssynbio.5b00124

Contributors

• Peng Geng
• Sean Leonard
• Kate Elston
• Dennis Mishler
• Jeffrey Barrick

Golden Gate Assembly

Golden Gate Assembly Links

Golden Gate Assembly - Start to Finish

• Tips, Tricks, and Troubleshooting Running into problems? Check out these potential solutions!

Kit-Specific Pages

• Yeast Toolkit (YTK) - Derived from Lee, Dueber 2015 (old Golden Gate Assembly page!)
• Broad Host range Toolkit (BTK) - Derived from Leonard, Barrick 2018

iGEM-Specific Protocols

• iGEM Alternate Part Plasmid Assembly

References

1. Lee ME, DeLoache WC, Cervantes B, Dueber JE. (2015) A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth. Biol. 4:975–986. Link
2. Engler C, Kandzia R, Marillonnet S. (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS ONE 3:e3647. Link
3. "Golden Gate Assembly". New England Biolabs. Retrieved 8 June 2015. Link
4. Iverson et al. (2016) CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology. ACS Syn Bio 5 (1), pp 99–103. http://pubs.acs.org/doi/abs/10.1021/acssynbio.5b00124

Contributors

• Peng Geng
• Sean Leonard
• Kate Elston
• Dennis Mishler
• Jeffrey Barrick

Golden Gate Assembly

Background & Design:
Golden Gate Assembly (GGA) was first described in Engler C, Kandzia R, Marillonnet S (2008) and Engler C, Gruetzner R, Kandzia R, Marillonnet S (2009) as an efficient way to quickly assembly multiple DNA sequences, or parts, into a single plasmid. This molecular cloning method allows a researcher to simultaneously and directionally assemble multiple DNA fragments into a single piece using Type IIs restriction enzymes and T4/T7 DNA ligase. This assembly is performed in vitro. Most commonly used Type IIs enzymes include BsaI, BsmBI and BbsI. Unlike standard Type II restriction enzymes like EcoRI and BamHI, these enzymes cut DNA outside of their recognition sites and therefore can create non-palindromic overhangs. Since 256 potential overhang sequences are possible, multiple fragments of DNA can be assembled by using combinations of overhang sequences.

Golden Gate Assembly Schematic:

Golden Gate Assembly Links

Golden Gate Assembly - Start to Finish

• Design a new part Design overhangs for the amplification of a new custom part to be assembled into a part plasmid.
• Assemble a new Part Plasmid Assemble a new custom part plasmid following amplification with part specific overhangs.
• Build a First-stage Plasmid (Transcriptional Unit) If you have all the requisite parts, you can build a complete plasmid with a single BsaI reaction.
• Build a Second-stage Plasmid Assemble multiple first-stage transcriptional unit plasmids.
• Tips, Tricks, and Troubleshooting Running into problems? Check out these potential solutions!

Kit-Specific Pages

• Yeast Toolkit (YTK) - Derived from Lee, Dueber 2015 (old Golden Gate Assembly page!)
• Broad Host range Toolkit (BTK) - Derived from Leonard, Barrick 2018

iGEM-Specific Protocols

• iGEM Alternate Part Plasmid Assembly

References

1. Lee ME, DeLoache WC, Cervantes B, Dueber JE. (2015) A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth. Biol. 4:975–986. Link
2. Engler C, Kandzia R, Marillonnet S. (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS ONE 3:e3647. Link
3. "Golden Gate Assembly". New England Biolabs. Retrieved 8 June 2015. Link
4. Iverson et al. (2016) CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology. ACS Syn Bio 5 (1), pp 99–103. http://pubs.acs.org/doi/abs/10.1021/acssynbio.5b00124

Contributors

• Peng Geng
• Sean Leonard
• Kate Elston
• Dennis Mishler
• Jeffrey Barrick

Golden Gate Assembly

Background & Design:
Golden Gate Assembly (GGA) was first described in Engler C, Kandzia R, Marillonnet S (2008) and Engler C, Gruetzner R, Kandzia R, Marillonnet S (2009) as an efficient way to quickly assembly multiple DNA sequences, or parts, into a single plasmid. This molecular cloning method allows a researcher to simultaneously and directionally assemble multiple DNA fragments into a single piece using Type IIs restriction enzymes and T4/T7 DNA ligase. This assembly is performed in vitro. Most commonly used Type IIs enzymes include BsaI, BsmBI and BbsI. Unlike standard Type II restriction enzymes like EcoRI and BamHI, these enzymes cut DNA outside of their recognition sites and therefore can create non-palindromic overhangs. Since 256 potential overhang sequences are possible, multiple fragments of DNA can be assembled by using combinations of overhang sequences.

Golden Gate Assembly Schematic:

Golden Gate Assembly Links

Golden Gate Assembly - Start to Finish

• Design a new part Design overhangs for the amplification of a new custom part to be assembled into a part plasmid.
• Assemble a new Part Plasmid Assemble a new custom part plasmid following amplification with part specific overhangs.
• Build a First-stage Plasmid (Transcriptional Unit) If you have all the requisite parts, you can build a complete plasmid with a single BsaI reaction.
• Build a Second-stage Plasmid Assemble multiple first-stage transcriptional unit plasmids.
• Tips, Tricks, and Troubleshooting Running into problems? Check out these potential solutions!

Kit-Specific Pages

• Yeast Toolkit (YTK) - Derived from Lee, Dueber 2015 (old Golden Gate Assembly page!)
• Broad Host range Toolkit (BTK) - Derived from Leonard, Barrick 2018

iGEM-Specific Protocols

• iGEM Alternate Part Plasmid Assembly

References

1. Lee ME, DeLoache WC, Cervantes B, Dueber JE. (2015) A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth. Biol. 4:975–986. Link
2. Engler C, Kandzia R, Marillonnet S. (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS ONE 3:e3647. Link
3. "Golden Gate Assembly". New England Biolabs. Retrieved 8 June 2015. Link
4. Iverson et al. (2016) CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology. ACS Syn Bio 5 (1), pp 99–103. http://pubs.acs.org/doi/abs/10.1021/acssynbio.5b00124

Contributors

• Peng Geng
• Sean Leonard
• Kate Elston
• Dennis Mishler
• Jeffrey Barrick

Golden Gate Assembly

Golden Gate Assembly Links

Golden Gate Assembly - Start to Finish

• Design a new part Design overhangs for the amplification of a new custom part to be assembled into a part plasmid.
• Assemble a new Part Plasmid Assemble a new custom part plasmid following amplification with part specific overhangs.
• Build a First-stage Plasmid (Transcriptional Unit) If you have all the requisite parts, you can build a complete plasmid with a single BsaI reaction.
• Build a Second-stage Plasmid Assemble multiple first-stage transcriptional unit plasmids.
• Tips, Tricks, and Troubleshooting Running into problems? Check out these potential solutions!

• Yeast Toolkit (YTK) - Derived from Lee, Dueber 2015 (old Golden Gate Assembly page!)
• Broad Host range Toolkit (BTK) - Derived from Leonard, Barrick 2018

iGEM-Specific Protocols

• iGEM Alternate Part Plasmid Assembly

References

Contributors

• Peng Geng
• Sean Leonard
• Kate Elston
• Dennis Mishler
• Jeffrey Barrick

Golden Gate Assembly

Background & Design:
Golden Gate Assembly (GGA) was first described in Engler C, Kandzia R, Marillonnet S (2008) and Engler C, Gruetzner R, Kandzia R, Marillonnet S (2009) as an efficient way to quickly assembly multiple DNA sequences, or parts, into a single plasmid. This molecular cloning method allows a researcher to simultaneously and directionally assemble multiple DNA fragments into a single piece using Type IIs restriction enzymes and T4/T7 DNA ligase. This assembly is performed in vitro. Most commonly used Type IIs enzymes include BsaI, BsmBI and BbsI. Unlike standard Type II restriction enzymes like EcoRI and BamHI, these enzymes cut DNA outside of their recognition sites and therefore can create non-palindromic overhangs. Since 256 potential overhang sequences are possible, multiple fragments of DNA can be assembled by using combinations of overhang sequences.

Golden Gate Assembly Schematic:

Supplies

• 10× T4 DNA ligase buffer (NEB: M0202T)
  • Why T4 DNA ligase buffer with T7 DNA ligase? T7 buffer contains PEG, which inhibits electroporation.
• T7 DNA ligase (NEB: M0318S)
  • Why T7 DNA ligase instead of T4 DNA ligase? T7 does not ligate blunt ended DNA, yielding less off-target ligation.
• Restriction endonuclease BsaI (NEB: R0535) or BsmBI (NEB: R0580S)
• Competent cells
• SOC and liquid media
• Selective plates

Protocol

Use the Calculator Spreadsheet (linked at the bottom of the page) for planning your reactions!

1. Mix 20 fmol (1 nM final concentration) backbone and 40 fmol each insert of your DNA segments together. The volume of this mixture must be 16 µL.
2. Add water to a final volume of 16 µl
3. Add 2 µL of 10× T4 DNA ligase buffer. Mix by vortexing.
4. Add 1 µL of BsaI or BsmBI and 1 µL of T7 DNA ligase. Mix by gently pipetting.
5. Incubate the reaction for 30 temperature cycles (42°C for 5 min and then 16°C for 5 min), followed by a final 10 min incubation at 55°C. Alternate cycling protocols are described below, and may show better assemblies in some situations.
6. Use 2 µL of this assembly reaction for electroporation or 4 µL for transforming chemically competent cells <- recommend change to 1uL.

1. Lee ME, DeLoache WC, Cervantes B, Dueber JE. (2015) A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth. Biol. 4:975–986. Link
2. Engler C, Kandzia R, Marillonnet S. (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS ONE 3:e3647. Link
3. "Golden Gate Assembly". New England Biolabs. Retrieved 8 June 2015. Link

• Peng Geng
• Sean Leonard
• Kate Elston
• Dennis Mishler
• Jeffrey Barrick

Golden Gate Assembly

Supplies

• 10× T4 DNA ligase buffer (NEB: M0202T)
  • Why T4 DNA ligase buffer with T7 DNA ligase? T7 buffer contains PEG, which inhibits electroporation.
• T7 DNA ligase (NEB: M0318S)
  • Why T7 DNA ligase instead of T4 DNA ligase? T7 does not ligate blunt ended DNA, yielding less off-target ligation.
• Restriction endonuclease BsaI (NEB: R0535) or BsmBI (NEB: R0580S)
• Competent cells
• SOC and liquid media
• Selective plates

Protocol

Use the Calculator Spreadsheet (linked at the bottom of the page) for planning your reactions!

1. Mix 20 fmol (1 nM final concentration) backbone and 40 fmol each insert of your DNA segments together. The volume of this mixture must be 16 µL.
2. Add water to a final volume of 16 µl
3. Add 2 µL of 10× T4 DNA ligase buffer. Mix by vortexing.
4. Add 1 µL of BsaI or BsmBI and 1 µL of T7 DNA ligase. Mix by gently pipetting.
5. Incubate the reaction for 30 temperature cycles (42°C for 5 min and then 16°C for 5 min), followed by a final 10 min incubation at 55°C. Alternate cycling protocols are described below, and may show better assemblies in some situations.
6. Use 2 µL of this assembly reaction for electroporation or 4 µL for transforming chemically competent cells <- recommend change to 1uL.

Golden Gate Assembly

Golden Gate Assembly - Start to Finish

• Design a new part Design overhangs for the amplification of a new custom part to be assembled into a part plasmid.
• Assemble a new Part Plasmid Assemble a new custom part plasmid following amplification with part specific overhangs.
• Build a First-stage Plasmid (Transcriptional Unit) If you have all the requisite parts, you can build a complete plasmid with a single BsaI reaction.
• Build a Second-stage Plasmid Assemble multiple first-stage transcriptional unit plasmids.
• Tips, Tricks, and Troubleshooting Running into problems? Check out these potential solutions!

Kit-Specific Pages

• Yeast Toolkit (YTK) - Derived from Lee, Dueber 2015 (old Golden Gate Assembly page!)
• Broad Host range Toolkit (BTK) - Derived from Leonard, Barrick 2018

iGEM-Specific Protocols

• iGEM Alternate Part Plasmid Assembly

References

1. Lee ME, DeLoache WC, Cervantes B, Dueber JE. (2015) A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth. Biol. 4:975–986. Link
2. Engler C, Kandzia R, Marillonnet S. (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS ONE 3:e3647. Link
3. "Golden Gate Assembly". New England Biolabs. Retrieved 8 June 2015. Link

Contributors

• Peng Geng
• Sean Leonard
• Kate Elston
• Dennis Mishler
• Jeffrey Barrick

This page serves as the main repository for everything Golden Gate Assembly-related. This page links to a number of protocol pages that will help you design DNA sequences for Golden Gate Assembly, learn about the various different Golden Gate Assembly techniques and when we employ each one, and conduct the different types of Golden Gate Assembly reactions.

Golden Gate Assembly (GGA) was first described in Engler C, Kandzia R, Marillonnet S (2008) and Engler C, Gruetzner R, Kandzia R, Marillonnet S (2009) as an efficient way to quickly assembly multiple DNA sequences, or parts, into a single plasmid.

List of Golden Gate Assembly subpages

• Yeast Toolkit (YTK) - Derived from Lee, Dueber 2015
• Broad Host range Toolkit (BTK) - Derived from Leonard, Barrick 2018
• Designing a new DNA part sequence
• Creating a part plasmid using BsmBI
• Creating a cassette plasmid using BsaI

Work in Progress. As of 12/14/2017, none of the above links are guaranteed to work or be accurate. This is a work in progress reorganization of the Barrick lab Golden Gate Assembly Protocols, including those used by the Microbe Hackers undergraduate research lab and UT Austin iGEM team.

-Dr. Mishler