DIVA DNA Construction
This capability relates to physical construction of sequence-validated plasmids. The workflow leverages laboratory automation to enable scale/reduce reagent costs, sequence validation provides full plasmid coverage, and is integrated with the DIVA/DeviceEditor/BOOST/j5 design, BLiSS biosecurity screening, and ICE sequence/strain repository platforms. This enables ABF researchers to focus their efforts where they are competitively advantaged (upstream and downstream of DNA construction). ABF operations are made more efficient through the pooling of DNA construction tasks across ABF researchers at scales that significantly benefit from laboratory automation devices. DNA fragments requiring de novo DNA synthesis are constrained by DNA synthesis vendor (e.g., Twist, ThermoFisher) capabilities. Extant physical DNA must be sequence validated before input (as digest substrate or PCR template) into the construction process. Each DNA assembly reaction may contain at most 5 assembly pieces (including the vector backbone); more than 6 assembly pieces requires a hierarchical (multi-step) construction process. PCR reaction size is capped at 5 kb; larger fragments need to be partitioned or a digest strategy may need to be pursued instead. Certain DNA assembly methods have specific requirements (e.g. lack of internal type IIs restriction sites). This capability is available internally to ABF researchers, as well as to ABF CRADA projects.
Lawrence Berkeley National Laboratory