GoldenGate Options
GoldenGate Options
Understanding GoldenGate Platforms: Key Terminology
To fully grasp how GoldenGate Cloning platforms function, it's important to understand a few key terms:
Multiplexing refers to one-pot reactions, where multiple fragments are digested and ligated simultaneously, rather than processing them separately with different restriction enzymes before annealing.
Assembly Organization determines how plasmids are cloned:
Serial assembly: Each new plasmid relies on prior work, meaning cloning occurs step by step.
Parallel assembly: Multiple plasmids can be cloned concurrently before being combined in later steps.
Cycling systems permit additional inserts using the final plasmid.
Non-cycling systems result in a dead-end plasmid, meaning further modular cloning requires modifications.
Overview of GoldenGate Modular Cloning Systems
Five primary GoldenGate modular cloning systems are covered here: Goldenbraid, Mobius, MoClo, GreenGate, and MultiGreen.
Each system has unique assembly structures, enzymes, and multiplexing capabilities. I have a summary table at the end of this section.
Of course, any of these systems can have more or fewer assembly elements, but generally, it is common practice to keep slots for certain elements as described by the platform so that plasmids made in multiple labs can be used together seamlessly. I.E. if someone in John Smith's lab uses "ATGA" and "GTAA" as the overhangs that flank promoters, then it would be more work for Jane Doe's lab to use any promoter from John Smith's lab since it doesn't have the "CTGA" and "ATCA" overhangs that flank promoters like the platform her lab uses (discalimer: I made up these overhangs)
Goldenbraid*
- Assembly size
- 3 components
- promoter - coding sequence(CDS) - terminator
- Enzymes
- BsaI and Esp3I
- Levels
- 0(Amp), A(Kan) & Ω(Spec)
- Multiplexing?
- <2 transcriptional units: parallel
- >2 transcriptional units: series
- Cycling?
- yes!
- Other notes:
- Unintuitive
- Unique terminology
- Need to transfer to binary
Mobius
- Assembly size
- 10-component assembly
- Distal -proximal - core - 5' UTR - N tag - CDS1 - CDS2 - C tag - 3 UTR - Terminator
- Enzymes
- BsaI and AarI
- Levels
- L0(Cm), L1(Kan), L2(Cm)
- Multiplexing?
- < 4 TU: Parallel
- >4 TU: Series
- Cycling?
- Yes
- Other notes:
- High learning curve, many moving pieces
- “Only” 4 acceptors
- Need to go through L1 to get to L2
- AarI and BsaI cut differently
- AarI is unlikely to be in assemblies by chance due to its longer recognition site, but it is also pretty pricey
- Pretty colors for selection - check out their usage of sfGFP and pink
- Need to transfer to binary
MoClo*
- Assembly size
- 5-component assembly
- Promoter - 5’ UTR - signal peptide - CDS -Term
- Enzymes
- BsaI and BpiI
- Levels
- L0(Amp), L1(Kan), L2(Spec)
- Multiplexing?
- < 7 TU: Parallel
- >7 TU: Series
- Cycling?
- Yes
- Other notes:
- BsaI and BpiI cut differently
- Need to transfer to binary
GreenGate*
- Assembly size
- 6-component assembly
- Promoter - signal peptide - CDS - signal peptide - Term - selection
- Enzymes
- BsaI
- Levels
- L0(amp), L1(Spec/Kan), L2(Spec/Kan)
- Multiplexing?
- No
- Cycling?
- No
- Other notes:
- Easy and good modularity
- Borders for Agro!
- No way to get multiple TUs easily
MultiGreen 2.0
- Assembly size
- 6-component assembly
- Promoter - signal peptide - CDS - signal peptide - Term - selection
- Enzymes
- BsaI
- Levels
- L0(amp), L1(Spec/Kan), L2(Spec/Kan)
- Multiplexing?
- Yes!
- Cycling?
- < 6 TU: Parallel
- >6 TU: Series
- Other notes:
- Made by my labmate
- I am biased, but it is great
- Borders for Agro!
- Allows for multi-level cloning
Summary:
The table below summarizes their differences:
| System | Assembly Size | Enzymes Used | Levels | Multiplexing? | Cycling? | Notes |
|---|---|---|---|---|---|---|
| GoldenBraid | 3 components (Promoter - CDS - Terminator) | BsaI, Esp3I | 0 (Amp), A (Kan), Ω (Spec) | <2 TU: Parallel, >2 TU: Serial | ✅ Yes | Unique terminology, requires binary transfer |
| Mobius | 10 components (Distal - Proximal - Core - 5' UTR - N Tag - CDS1 - CDS2 - C Tag - 3' UTR - Terminator) | BsaI, AarI | L0 (Cm), L1 (Kan), L2 (Cm) | <4 TU: Parallel, >4 TU: Serial | ✅ Yes | High learning curve, needs L1 transition, color-based selection |
| MoClo | 5 components (Promoter - 5’ UTR - Signal Peptide - CDS - Terminator) | BsaI, BpiI | L0 (Amp), L1 (Kan), L2 (Spec) | <7 TU: Parallel, >7 TU: Serial | ✅ Yes | Requires binary transfer |
| GreenGate | 6 components (Promoter - Signal Peptide - CDS - Signal Peptide - Terminator - Selection) | BsaI | L0 (Amp), L1 (Spec/Kan), L2 (Spec/Kan) | ❌ No | ❌ No | Easy modularity, lacks multi-TU capability |
| MultiGreen 2.0 | 6 components (Same as GreenGate) | BsaI | L0 (Amp), L1 (Spec/Kan), L2 (Spec/Kan) | ✅ Yes | <6 TU: Parallel, >6 TU: Serial | Supports multi-level cloning, borders for Agrobacterium |
So how do I choose?
What does your lab do? – do you need modular cloning?
- If you primarily work with guides and use the same system, Gateway or Type II cloning may suffice
- GreenGate and MultiGreen
<3 → GoldenBraid
4-6 → MoClo, GreenGate, MultiGreen
>6 → Mobius
How
many TU do you need per final plasmid?
- 2 → GoldenBraid
- 4 → Mobius
- 6 → GreenGate/MultiGreen
- 1 → MoClo
Do
you need multiple transcriptional units?
- GoldenBraid, MoClo, MultiGreen, Mobius
- Mobius, MoClo, MultiGreen
- Mobius, MoClo, MultiGreen
*It is worth noting that many of these platforms have undergone updates that address some of the problems I list here. I didn't have time to address every update, so I kept to the published platforms. However, here are the updates for each system I could find:
Alternative Option
Another modular cloning approach is GAANTRY, which is recombinase-based, builds directly into Agrobacterium, and skips the E. coli transformation step.
GoldenGate Options for Plant Science
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