Barrick Lab :: GoldenGateAssemblyProtocolsMainPage

---+ Golden Gate Assembly
 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.

*Background & Design*: <br /> Golden Gate Assembly (GGA) was first described in [[http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0003647][Engler C, Kandzia R, Marillonnet S (2008)]] and %BLUE%<u>[[http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0005553][Engler C, Gruetzner R, Kandzia R, Marillonnet S (2009)]]</u>%ENDCOLOR% 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:*

<img width="712.5" alt="goldengatediagram3.png" src="/twiki/pub/Lab/GoldenGateAssemblyProtocolsMainPage/goldengatediagram3.png" height="345.6" />

---++ *Golden Gate Assembly Links*

*Golden Gate Assembly - Start to Finish*
   * [[Protocols BTK Design a new part][Design a new part]] Design a new part with correct part-type overhangs.
   * [[Protocols BTK Make a new part plasmid][Assemble a new part plasmid]] Assemble a new part plasmid.
   * [[Protocols BTK Assemble single TU plasmid][Build a first-stage plasmid (Transcriptional Unit, BsaI assembly)]] Build a complete plasmid with a single BsaI reaction.
   * [[Protocols BTK Assemble multiple TU plasmid][Build a second-stage plasmid]] Assemble multiple first-stage transcriptional unit plasmids.
   * [[Protocols Golden Gate Troubleshooting][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: [[%ATTACHURL%/BroadHostRange_Reaction_Calculator.xlsx][BroadHostRange_Reaction_Calculator.xlsx]]
   * NEBeta Tools for the design of high-fidelity Golden Gate Assemblies: https://www.neb.com/research/nebeta-tools

*iGEM-Specific Protocols*
   * [[Protocols Golden Gate Making A New Part][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&ndash;986. [[http://pubs.acs.org/doi/abs/10.1021/sb500366v][Link]]
   1 Engler C, Kandzia R, Marillonnet S. (2008) A one pot, one step, precision cloning method with high throughput capability. _PLoS ONE_ *3*:e3647. [[http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0003647][Link]]
   1 "Golden Gate Assembly". New England Biolabs. Retrieved 8 June 2015. [[https://www.neb.com/applications/cloning-and-synthetic-biology/dna-assembly-and-cloning/golden-gate-assembly][Link]]
   1 %BLACK%Iverson et al. (2016) CIDAR Mo%ENDCOLOR%Clo: 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&ndash;103.%BLACK% %ENDCOLOR% http://pubs.acs.org/doi/abs/10.1021/acssynbio.5b00124

---++ *Contributors*
   * Peng Geng
   * Sean Leonard
   * Kate Elston
   * Dennis Mishler
   * Jeffrey Barrick
---
 -- Main.KateElston - 29 Jan 2018

---++The excel worksheet for calculating Golden Gate Assembly Reactions can be found in the attachment below.
Attachments
Topic attachments
I	Attachment	History	Action	Size	Date	Who	Comment
xlsx	BroadHostRange_Reaction_Calculator.xlsx	r6 r5 r4 r3 r2	manage	17.0 K	2021-07-09 - 20:54	KateElston	Golden Gate Reaction Calculator
ai	golden_gate_diagram.ai	r1	manage	389.9 K	2018-01-30 - 17:16	KateElston
This topic: Lab > WebHome > ProtocolList > GoldenGateAssemblyProtocolsMainPage
Topic revision: r14 - 2021-07-12 - KateElston