![]() (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (A) Equimolar amounts of BsaI or BsmBI deprotected 13FNIII synthons were incubated with 1 unit of T4 ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). Final assemblies ( 3a and 4) contain 5′-BamHI and 3′-NotI restriction sites for transfer into expression vectors.Įfficient synthon assembly with split-and-pool reactions. pDA-N vectors that carry synthons encoding amino-terminal elements of protein constructs ( 1) can be combined with synthons encoding carboxyl-terminal elements from pDA-C vectors ( 2) to yield a composite product ( 3a), optionally leaving an additional entry point via BsaI restriction sites to insert additional synthons ( 3b). (B) For applications that require modular recombination or insertion of individual elements, synthons can be ligated into the modular assembly vectors pDA-N or pDA-C. (A) Deprotected synthons can be ligated into a BsaI-digested shuttle vector (pShuttle) that contains 5′-BamHI and 3′-NotI restriction sites compatible with our in-house collection of expression vectors. Vectors for synthon recombination and transfer to expression plasmids. The same strategy can be applied to the assembly of heterosynthons (dashed box), which allows for the engineering of chimeric and multimodular proteins or polycistronic genes.īuffer and temperature preferences of the BsmBI/BsaI/BsmAI system. Each product module can recursively enter the assembly cycle (left panel) N times to yield concatameric synthons with 2N elements. Pooling of the deprotected synthons in the presence of ligase results in unidirectional assembly, affording an idempotent tandem repeat synthon by restoration of orthogonal protecting groups on opposite ends. Cohesive ends of entry synthons are selectively deprotected by digestion with either BsaI or BsmBI. ![]() (B) Schematic representation of the ‘split-and-pool’ assembly principle. 5′-xxxx) at one end of the synthon, while maintaining orthogonal protection groups (with 5′-YYYY vs. Restriction by either BsaI or BsmBI selectively exposes user-definable 4-base cohesive overhang sequences (5′-XXXX vs. (A) Entry synthons are flanked on both sides by recognition sequences for the type IIS endonucleases BsaI and BsmBI. ![]()
0 Comments
Leave a Reply. |