EENdb - Utilities  >  TALE construction methods
This page describes and compares the TALEN/TALE construction methods.
For web-tools and resources of TALE/TALEN engineering, see the page of Engineering Resource.

List of TALE construction methods
Golden Gate cloning-based assembly GG
(Golden Gate)
Golden Gate (vector based) method uses type IIS restriction enzymes to create multiple sticky ends, and ligate 8-10 TALE repeats/fragments in one reaction.

Five groups developed GG with different details and EENdb distinguish them by the corresponding author(s): GG(L), GG(Y), GG(V&B), GG(M), GG(B&B).
> Quick, about 1 week.
> Need many pre-made plasmids.
> Rely on sequencing to identify the correct clone.
21421566 (L)
21459844 (Y)
21493687 (V&B)
21625552 (M)
21625585 (B&B)

EENs by GG
(Golden Gate - PCR based)
(Named by EENdb)
Golden Gate (PCR based) method is a modified Golden Gate method which uses PCR fragments instead of plasmid fragments. 4 or 6 TALE repeats/fragments are ligated in one reaction. > Quicker than GG; 3-4 days.
> Need some pre-made plasmids (less than GG) and primers.
> Rely on sequencing to identify the correct clone.
21248753 (original)
22222791 (improved protocol)

Sequential assembly UA
(Unit Assembly)
Unit Assembly method uses a strategy similar to BioBrick in synthetic biology, which is a step-by-step module-built method using the same restriction endonucleases for all the steps. > Need one weeks starting from 4 single unit modules; or 3 days from pre-made 1- to 4-unit modules.
> Based on regular molecular-cloning techniques with simple starting plasmids.
> Vectors from all the intermediate steps could be saved and re-used to speed up construction.
> No restriction on the number of TALE repeats.

EENs by UA
(Restriction Enzyme And Ligation)
REAL and REAL-Fast methods are based on type IIS restriction enzymes. About 4 different types of sticky ends and 4 different groups of modules are used (additional start and end groups are also needed).
Single modules are used in REAL and pre-assembled 2- to 4-unit modules are used in REAL-Fast (and also in FLASH; see below).
> Need 1 weeks.
> Need many pre-made plasmids and primers.
21822241 (first report)
23026907 (protocol)

High-throughput solid-phase assembly FLASH
(Fast Ligation-based Automatable Solid-phase High-throughput)
FLASH method is derived from REAL-Fast and based on solid-phase synthesis.
The same 2- to 4-unit modules as used in REAL-Fast are employed.
> High-throughput, more than 96 TALE arrays can be assembled within one day.
> Need special equipments and reagents as well as many pre-made plasmids.
> Rely on sequencing to identify the correct clone.

(Iterative Capped Assembly)
ICA method is based on solid-phase synthesis.
Single unit modules generated by type IIS restriction enzymes with 4 different types of sticky ends are employed.
In each step, harpin capping oligos are added to inactivate incomplete (unligated) chains in the previous step, in addition to the modules for enlongation.
> High-throughput.
> Need special equipments and reagents.


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