January 2020 welcomed a new kid in the Cloning Block, and yes it seems to be pretty promising! It is an advanced form of the #GibsonAssembly and of course is #homologybased. It uses a simple enzyme mix of the restriction enzyme, #SfiI and #T5Exonuclease and in ~30 mins you have your coveted 'halo' ( i mean cloned #plasmid!)
Principle
SfiI enzyme : This utilizes #SfiI which is a Type-2 restriction #endonuclease that has the restriction site:
GGCCNNNN | NGGCC CCGGN | NNNNCCGG
It is a relatively rare cutting enzyme with the recognition sites occurring at low frequencies in the A. thaliana genome (~3 sites per million base pairs) in contrast to the 265 BsaI sites per million base pairs.
T5 Exonuclease: The other special component used is the #T5Exonuclease which actually is a #dsDNA specific exonuclease and #ssDNA endonuclease aka #flapendonuclease. The TEDA: T5 Exonuclease Dependent Assembly is a fascinating technique in itself where the enzyme chews up 5' ends of linearized plasmids and insert DNA stretches only for the 5' overhangs to anneal specifically between them.
Protocol
The technique revolves around preparing an entry vector with 2 specific and distinct adapter sequences and #SfiI cut sites, and a destination vector that has also been modified to include the adapter and SfiI cut sites.
FIGURE: #EntryVector and #DestinationVector designs for #NimbleCloning.
FIGURE: Schematics of the #digestion and #recombination
The Adapter sequences were computationally created to be resistant to restriction endonuclease cut and unique. Mix your plasmids, enzymes and sprinkle #PEG8000, #DTT, #Tris and #MgCl2, cook for ~30mins at 50 C, and you are ready to feed it to your little competent cells!
Merits
1. In Nimble, assembly accuracy is high, enabling the simultaneous assembly of multiple parts in a single-tube reaction.
2. It uses two circular plasmids to clone a fragment using a restriction enzyme, no need to linearize plasmids unlike Gibson Assembly.
3. Nimble uses unique nucleotide sequences or adapters enabling the use of standardized primers, whereas Gibson Assembly or In Fusion does not have standardized primer design methods.
4. Gateway cloning leaves a 25bp scar at the recombination site, whereas Nimble can be scar-less if the primers are designed differently. The T5 exonuclease can also degrade the SfiI sequence as well as the 21bp adapters.
5. The T5 exonuclease can degrade the denatured, nicked or linear DNA which may be present in the plasmid sample used, and can cleanup the incomplete ligation products even.
6. Lastly Nimble is being considered a bit cheaper than its contemporaries.
For you to gulp:
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