Figure 1.
Split-and-pool assembly of DNA synthons.
(A) Entry synthons are flanked on both sides by recognition sequences for the type IIS endonucleases BsaI and BsmBI. Restriction by either BsaI or BsmBI selectively exposes user-definable 4-base cohesive overhang sequences (5′-XXXX vs. 5′-xxxx) at one end of the synthon, while maintaining orthogonal protection groups (with 5′-YYYY vs. 5′-zzzz overhangs) at the opposite end. (B) Schematic representation of the ‘split-and-pool’ assembly principle. Cohesive ends of entry synthons are selectively deprotected by digestion with either BsaI or BsmBI. 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. Each product module can recursively enter the assembly cycle (left panel) N times to yield concatameric synthons with 2N elements. 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.
Table 1.
Buffer and temperature preferences of the BsmBI/BsaI/BsmAI system.
Figure 2.
Vectors for synthon recombination and transfer to expression plasmids.
(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. (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. 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). Final assemblies (3a and 4) contain 5′-BamHI and 3′-NotI restriction sites for transfer into expression vectors.
Figure 3.
Efficient synthon assembly with split-and-pool reactions.
(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). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of (13FNIII)1 to (13FNIII)8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the (13FNIII)8 construct.
Figure 4.
Expression and characterization of 13FNIII and VWFA2 tandem repeat proteins.
Superposed elution profiles from size exclusion chromatography of (13FNIII)2–8 proteins (A) and (VWFA2)6–10 (B). (C,D) Coomassie stained SDS-PAGE of the purified proteins. (E,F) Unfolding curves from Thermofluor analysis suggest that the concatameric constructs are properly folded. Note the consistent shift of the (VWFA2)n unfolding curves in the presence and absence of Ca2+.
Table 2.
Apparent melting temperature of concatameric proteins.
Figure 5.
Assembly of chimeric constructs and one-pot concatamer formation.
(A) Assembly of (FNIII)2-VWFA2-(FNIII)2 sandwich constructs from a modular assembly vector (top). PCR amplification with specific primers (indicated above the lanes; Table S1) show that the A2 synthon is sandwiched between two 13FNIII repeats. (B) One-pot concatamer formation with orthogonal chain stoppers. Fully deprotected synthons are mixed in different molar ratios with orthogonal chain stoppers (equivalent synthons with protecting groups on one end). Increasing the concentration of chain stoppers shifts the size distribution towards shorter concatamers. Molar ratios of unprotected synthons:chain stoppers are indicated at the top of the lanes. Bands marked with asterisks presumably correspond to circularized dimers.