GoldenGate Overview

What it isn't

If you've heard of Gateway Cloning, you might wonder how it compares to GoldenGate Cloning—but they are fundamentally different. Gateway Cloning relies on recombinases (the same principle behind the GAANTRY system), whereas GoldenGate uses Type II enzymes for its assembly process. 

What it is

So, how do GoldenGate reactions work? To simplify:

• Entry vectors (Level 0 plasmids) are digested using a Type II enzyme (such as BsaI)
• The overhangs generated during digestion enable unidirectional assembly when the fragments are ligated

Where to Buy GoldenGate Cloning Materials

GoldenGate cloning kits are available from multiple providers. We use New England Biolabs (NEB), and below is a breakdown of kit options:

Kit Name	Includes BsaI?	Price per Reaction
NEBridge Golden Gate Assembly Kit (BsaI -HF v2)	✅ Yes	$8.90
NEBridge Ligase Master Mix	❌ No	$1.94 + ~$0.80 for BsaI

Keep in mind that these kits only include the necessary reagents—you’ll still need to select a cloning system(aka platform) to use. We’ll discuss these options later on.

Additionally, NEB provides an online tool for in silico GoldenGate assembly without the need for any software: NEBridgeGoldenGate Assembly Tool.

GoldenGate Assembly Parameters

A typical GoldenGate reaction requires:

• The appropriate DNA fragments

• A ligase and buffer

• A Type IIS restriction enzyme

All components are combined in a one-pot reaction, and the thermocycler is programmed to alternate between digestion (16°C) and ligation (37°C). After 30 cycles, the assembled construct should be present, albeit in small amounts.

What Happens After Assembly?

Once assembly is complete, the reaction is transformed into E. coli to amplify the plasmid.

But what should you expect on your plate after transformation?

• Partial assemblies or lone digested fragments—since these are not plasmids with origins of replication (oris), E. coli won't multiply them.

• Undigested or reassembled backbones or entry vectors—most systems use CCDB/suicide gene selection, minimizing this concern.

• Entry vectors with different selection markers—since entry vectors often use Amp resistance while the acceptor plasmid uses Kan/Spec, entry vectors shouldn't survive on plates meant for the acceptor.

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Expected Outcome

Ideally, your correctly assembled construct:

• Eliminates the recognition site for the Type II enzyme

• Integrates into the correct backbone/acceptor plasmid, allowing for E.coli replication

• Confers antibiotic resistance, enabling passive selection in E. coli

Of course, biology is unpredictable, and unexpected outcomes may arise, but these guidelines help minimize errors.

Screening and Quality Checking

Of course, biology is unpredictable, and unexpected outcomes may arise, but these guidelines help minimize errors.
To ensure successful cloning:

✅ Always check for both the insert and backbone when screening

✅ Use proper antibiotic selection for the backbone/acceptor

✅ Digest your prep before sequencing

✅ Sequence your construct whenever possible

Sidenote, in Mobius, we use visual selection instead of toxic CCDB, allowing plasmids to visually indicate whether they have accepted the insert.

Modular Cloning Jargon and Concepts

Restriction Enzymes for Modular Cloning

GoldenGate Overview

GoldenGate Options for Plant Science

Setup and Staying Organized

Misc. Notes