GoldenGate compatibility

GoldenGate assembly is an assembly method based off of using Type IIS restriction enzymes. It allows for high efficiency cloning of DNAs in a modular fashion. Because it is still restriction enzyme based, sites need to be taken out of parts that will be assembled. However, parts can be put into plasmids for modular assembly, an advantage over gibson assembly and related techniques. Unlike BioBrick assembly, many parts can be put together at once.

Usual enzymes include BsaI and BsmBI. Below is an example of a 5 part 1 vector assembly.

I-SceI_GG-History

Popular methods for GoldenGate include MoClo and GoldenBraid. Since these methods normally require many premade vectors, the base vector only supports insertion between 2 BsmBI sites derived from the base vector from the Yeast Toolkit. Parts can also be converted into GoldenGate compatible parts using SLiCE.

Addgene gives a good overview of exactly how GoldenGate works.

 

Enzymes:

BsaI- Use BsaI rather than BsaI-HF. BsaI apparently has better function with the T4 buffer at a higher temperature

BsmBI- More expensive than BsaI, also works at a higher temperature. Possibly better in the final cut reaction(?)

BbsI- Apparently needs storage at -80. Cuts 2bp away from recognition site instead of 1bp like BsaI or BsmBI

BtgZI- Cuts 10bp away from recognition site. Can be used to jump over BsaI. Extremely common in E coli genome

AarI/ (BfuAI, BspMI) – AarI is a 7bp cutter and includes BfuAI and BspMI. AarI is also extremely expensive, so avoiding this enzyme in modular cloning reactions is advisable

SapI – 7bp cutter. Cuts for overhang of only 3bp. Used in the Electra cloning system.