GoldenGateAssembly < Lab

---+ Golden Gate Assembly

Golden Gate Assembly is a [[https://en.wikipedia.org/wiki/Molecular_cloning][molecular cloning]] method used to recombine multiple DNA components into a single linear piece or plasmid. It bears similarity to [[https://blog.addgene.org/plasmids-101-restriction-cloning][restriction cloning]] by using a restriction endonuclease to produce "sticky ends", allowing for two terminal ends of DNA (either two distinct DNA molecules or a single linear DNA molecule circularizing) to hybridize and be annealed together via a DNA Ligase. 

To perform Golden Gate Assembly, one or more Type IIS Restriction Enzymes are used. Commonly used enzymes in the Barrick Lab are:

   * [[https://www.neb.com/products/r0739-bsmbi-v2][BsmBI-V2]] - Recognizes the 6-base long 'CGTCTC'. Produces a four base overhang beginning after one base downstream of the recognition site. Active at 42°C.
      * NEB produces [[https://www.neb.com/products/e1602-nebridge-golden-gate-assembly-kit-bsmbi-v2][Golden Gate Assembly kits]] for this enzyme.
   * [[https://www.neb.com/products/r3733-bsai-hf-v2][BsaI-HFV2]] - Recognizes the 6-base 'GGTCTC'. Produces a four base overhang site beginning one base downstream of the recognition site. Active at 37°C.
      * NEB produces [[https://www.neb.com/products/e1601-nebridge-golden-gate-assembly-mix-basai-hf-v2][Golden Gate Assembly kits]] for this enzyme.
   * [[https://www.neb.com/products/r0745-paqci][PaqCI]] - Recognizes the 7-base long 'CACCTGC' sequence. Produces a four base overhang beginning after four bases downstream of the recognition site. Requires an additional included "activator" reagent. Active at 37°C.
      *   PaqCI is particularly useful in cases when components are not otherwise domesticated as the longer recognition sequence reduces the likelihood of undesired cut sites.
   * [[https://www.neb.com/products/r0569-sapi#Product%20Information][SapI]] - Recognizes the 7-base long 'GCTCTTC' sequence. Produces a *three* base overhang beginning after one base downstream of the recognition site. Active at 37°C.
      * Only recommended when other enzymes have undesired cut sites.

All reactions require T4 DNA Ligase Buffer. Unless you are using a preprepared Golden Gate Assembly kit such as NEBridge, you will also need [[https://www.neb.com/products/m0202-t4-dna-ligase][T4 DNA Ligase]].

----+ Preparation of DNA for Golden Gate Assembly

Golden Gate Assembly is not strict about the source of DNA used. It is advisable to proceed either from domesticated (cleansed of unwanted cut sites) plasmids or PCR products. Modular domesticated plasmids offer increased scalability and storage advantages while PCR offers the ability to introduce otherwise nonexistant recognition sites. For examples of each, see the [[https://barricklab.org/twiki/bin/view/Lab/BroadHostRangeToolkit][Bee Tool Kit]] and [[https://barricklab.org/twiki/bin/view/Lab/ProtocolsAcinetobacterGoldenTransformation][Acinetobacter Golden Transformation]], respectively.

Exactly how to produce overhangs varies between experiments and is not described here in detail. If you are using a set of pre-domesticated plasmids, you may have a toolkit worth referencing that discusses overhangs (such as the Bee Tool Kit). Pre-described methods that utilize linear fragments may also discuss overhangs (such as Acinetobacter Golden Transformation). Several tools exist to help you prepare and double check your custom designed fragments with your own overhangs. For example:

   * [[https://www.benchling.com/blog/golden-gate-assembly-wizard][Benchling's Golden Gate Assembly Wizard]] assists in fragment selection and primer design.
   * [[https://goldengate.neb.com/#!/][NEBridge Golden Gate Assembly Tool]] is similar to Benchling's without requiring an account.
   * [[https://pubs.acs.org/doi/10.1021/acssynbio.8b00333][Potapov et. al. 2018]] describes the most efficient overhangs.

Be sure to inspect your assemblies _in silico_ prior to performing the assembly to ensure that inserts are in the correct orientation and location relative to other components of your assembly.

After producing your fragments, calculate the appropriate quantity to use in your reaction. NEB advises that you should use no more than 75ng of DNA. Many online sources suggests using a 3:1 molar ratio between your insert and your backbone; however, what is an 'insert' and what is a 'backbone' can often be ambiguous. Reactions should proceed at high enough efficiency with a 1:1 molar ratio across all DNA components. Consider increasing the relative concentration of low molecular weight (short) DNA fragments to 3:1 or 5:1 ratios relative to your largest component.

---+ Preparing the Reaction Mix

Using NEB supplied reagents, there are two approaches to preparing the reaction mixtures. The first is to use prepared enzyme mixtures supplied by NEB. These mixtures contain enzyme and ligase in appropriate ratios, reducing errors pipetting, ensuring enzymes have been treated equally, and ensuring that all components (except DNA) are of equal age. These are only available using BsmBI-V2 and BsaI-HFV2. The other approach is to prepare mixtures by sourcing the restriction enzyme and T4 DNA ligase separately. This provides more control over reaction conditions at the cost of increased preparation complexity and uncertainty of reagent quality.

---+++ Preparing Golden Gate Assembly Mixes Using NEBridge (Beginner Friendly)

The Barrick Lab recommends using NEBridge kits (linked above for their respective enzymes) when the DNA fragments that will compose the completed assembly are determined not to contain unintended cut sites. The following protocol is derived from [[https://www.neb.com/protocols/2020/01/15/golden-gate-assembly-protocol-for-using-neb-golden-gate-assembly-kit-bsmbi-v2-neb-e1602][the instructions provided by NEB]] on February 22nd, 2023.

| *Reagent* | *Quantity* | *Notes* |
| DNA| As much as 6 &#956;l| Up to 75ng DNA|
| T4 DNA Ligase Buffer (10X) | 1 &#956;l | |
| NEB Golden Gate Enzyme Mix | 0.5–1 &#956;l | Use higher range for > 10 fragments |
| Nuclease-free H2O | to 10 &#956;l | A lower volume is used to save reagents. Scale the reaction in a mastermix or scale up to 20 &#956;l for low throughput reactions to avoid pipetting error |

---+++ Preparing Golden Gate Assembly Mixes Piecemeal (Intermediate)

The following protocol is derived from [[https://www.neb.com/protocols/2021/01/11/golden-gate-assembly-protocol-using-paqci-neb-r0745-and-t4-dna-ligase-neb-m0202][the instructions provided by NEB for PaqCI]] on February 22nd, 2023.

| *Reagent* | *Quantity* | *Notes* |
| DNA| As much as 6 &#956;l| Up to 75ng DNA|
| T4 DNA Ligase Buffer (10X) | 1 &#956;l | Do not use rCutsmart or other buffers that come with your restriction enzyme |
| Restriction Enzyme | 0.25–1 &#956;l | Use higher range for > 10 fragments |
| T4 DNA Ligase | 0.25–1 &#956;l | Use higher range for > 10 fragments |
| (PaqCI Activator) | 0.25–2 &#956;l | For use only with PaqCI as a restriction enzyme. Use higher range for > 10 fragments |
| Nuclease-free H2O | to 10 &#956;l | A lower volume is used to save reagents. Scale the reaction in a mastermix or scale up to 20 &#956;l for low throughput reactions to avoid pipetting error |

This is similar to the recommended volumes given by NEB, but with a volume scaled down by 50%. We find that scaling to save reagents still performs well, especially when using commercially-prepared competent cells that have high transformation efficiency. Exercise caution and consider scaling up the reaction to the recommended 20&#956;l total volume if you are not performing multiple reactions in parallel with a master mix. [[https://www.pipettipfinder.com/Topics/Why-We-Dont-List-Pipettor-Accuracy.aspx][Pipetting with small-volume pipettes is less accurate]]. Preparing reagents (except for source DNA that is unique per-reaction) in a master mix allows you to increase pipetting volume and therefore accuracy. 

----+ Thermocycling Conditions

[[https://www.neb.com/en-us/-/media/nebus/files/application-notes/technote_breaking_through_the_limits_of_golden_gate_assembly.pdf?rev=d8ac2ce3ef664c6c875e126d86153ec1&hash=A9F149CB12CEF2D48ECB6D175250282D][NEB documentation]] shows that efficiency and accuracy of assembly approves up to 60 cycles (using 5 minute steps) with diminishing returns past 15 cycles for accuracy. Generally speaking, the more cycles the better. In house, we use either 30 cycles for same-day protocols or as high as 90 cycles for overnight runs, especially with higher numbers of fragments. We also recommend a final hold of 12°C. This is plenty sufficient for short term storage while reducing ware on equipment compared to 4°C.

As such, we recommend the following thermocycling conditions, largely following NEB:
   * 5 minute incubation at 37°C or 42°C, depending on the enzyme employed, for cutting
   * 5 minute incubation at 16°C for ligation
   * Repeat the above 2 steps for a total of 30-90 cycles, as time allows
   * 10 minute incubation at 60°C to kill the active enzymes
   * Hold at 12°C until retrieval

---+ Citations

If you use Golden Gate Assembly in your research, you should site [[http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0003647][Engler C, Kandzia R, Marillonnet S (2008)]] and [[http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0005553][Engler C, Gruetzner R, Kandzia R, Marillonnet S (2009)]]

---+ See also
   * [[Generating Overhangs][Generating Overhangs]]
   * [[Broad Host Range Toolkit][Bee Microbiome Tool Kit]]

---++ Comments

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Barrick Lab > ProtocolList > GoldenGateAssemblyProtocolsMainPage > GoldenGateAssembly
Topic revision: r5 - 2024-05-21 - 18:47:40 - Main.CameronRoots