Table 1.

Comparison of Multisegment DNA Assembly Methods

	Gateway	Biobricks	Gibson	Yeast HR	LCR
Days to Completiona	4	3	3	5	3
Hands-0n Time (hrs)b	1–2	1–2	1–2	1–2	1–2
Premade Partsc	yes	yes	no	no	no
Scarless	no	noi	yes	yes	yes
Restriction Site Dependentd	no	yes	no	no	no
Construction Over 30kbpe	no	no	yes	yes	yes
Edit Existing Assemblyf	no	no	no	yes	no
Additional Ordered Assemblyg	no	yes	no	yes	yes
Costh	$$$	$	$	$	$
Commercial Kits Available	yes	yes	yes	no	no

^aTime in days for a 3-segment assembly (two inserts plus a vector) starting with PCR segments or restriction fragments and ending with an assembled plasmid purified from E. coli.

^bTotal bench time. Total time pipetting at the bench, including reaction set up and transformation into yeast or E. coli. Does not include overnight growth of yeast and E. coli, incubation times or sequencing. Assumes some expertise in conventional cloning.

^cBiobricks and Gateway require premade parts or entry plasmids that significantly increase the time to final assembly. Since the parts or entry plasmids can be reused, this extra time is only required for the first unique assembly.

^dLimited by restriction enzyme recognition sites. Sites found within segments need to be mutated before assembly.

^eConstructions over 30kpb will result in adequate efficiency and accuracy.

^fPossible to make targeted changes to a single element of a construction. This is most easily accomplished with yeast HR though a unique restriction site somewhere within the element is necessary.

^gEach assembly can be used to generate additional ordered assemblies by the same method. One example would be combining multiple expression cassettes (transcription units) in order to generate a synthetic metabolic pathway.

^hUp front and consumable supply cost.

ⁱGolden Gate can be quasi-scarless, see text