Description
This is Smithy. A web application that provides design automation for complex DNA assembly experiments. Smithy serves to aid researchers in their efforts to correctly build the gene assemblies they expect, helping them answer the fundamental questions, "Where can I find my parts, and how can they be put together?"
Through a simple input interface, complete insert and backbone sequences are provided for the design process. Along with the DNA sequences for an assembly the cloning method to use, sequence repositories for parts sourcing (AddGene, DNASU, iGEM, etc), and general experimental parameters (monovalent ion, divalent ion, dNTP concentrations, etc) are collected. Smithy uses these inputs to generate several complete assembly insert designs that:
- have minimized sets of sub-sequences for an assembly,
- source and plasmid information for every sub-sequence,
- identification of sub-sequences that must be directly synthesized,
- have a set of primers that facilitate assembly,
- have thorough thermodynamic analysis for each primer provided by Primer3-py,
- are in accordance with an researcher's experimental environment.
Cloning Methods
The cloning methods Smithy currently supports are Golden Gate, overlap extension based methods, and Gibson Assembly. Before an assembly design is created one of these cloning methods is selected so that Smithy can properly design primers for each sub-sequence used to assemble a researcher's desired insert.
Sequence Repositories
Smithy utilizes local copies of popular sequence repositories, saved in BLAST databases, for BLAST queries of the assembly's insert. The currently supported repositores are AddGene, DNASU, and iGEM. All sequences provided by these repositories are accessible by Smithy's query functionality. An insert sequence is queried agaist these local sequence databases using BLAST's nucleotide query method and the results are used to create complete assembly-ready sets of sequences for a cloning experiment.
Assembly Parameters
The essential parameters for a cloning experiment are gathered at the input. These parameters are:
- monovalent and divalent ion concentrations
- dNTP concentrations
- DNA sequence concentrations
- melting temperature
Additional parameters used by Smithy are:
- overlap amount (for overlap-based assemblies only)
- overhang count (for Golden Gate cloning only)
- user's selection of BLAST databases to use
- minimum size (in nucleotides) of BLAST query alignment results to use from each database
- maximum size (in nucleotides) of BLAST query alignment results to use from each database
- minimum size (in nucleotides) of synthetized sequences a user would purchase for an experiment
- maximum size (in nucleotides) of synthetized sequences a user would purchase for an experiment
All of the parameters listed above will be asked for at the input stage for a new assembly.