Part:BBa_I51001:Design
BioBrick base vector, as revised by Codon Devices
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 1327
Illegal suffix found in sequence at 1 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1327
Illegal NheI site found at 177
Illegal NheI site found at 1126
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NotI site found at 9
Illegal NotI site found at 1333 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1327
Illegal BglII site found at 1783 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 1327
Illegal suffix found in sequence at 2 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 1327
Illegal XbaI site found at 1342
Illegal SpeI site found at 2
Illegal PstI site found at 16 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1671
Design Notes
Synthesis of the BioBrick base vector design (BBa_I51000) was problematic. We encountered two issues while trying to construct the base vector. First, troubleshooting efforts during synthesis compromised the design of our parts: failed attempts to clone the base vector into an E. coli strain intolerant of expression of the toxic protein CcdB led to an unnecessary redesign of the ccdB positive selection marker in the BioBrick base vector. As a result, a revised design for the BioBrick base vector (BBa_I51001) was specified instead. Second, faulty part design adversely impacted the synthesis process: our pUC19-based replication origin design (BBa\_I50020) was similarly nonfunctional, so the revised base vector (BBa_I51001) could not be propagated as specified. We eventually determined that the provided DNA for the BioBrick base vector was actually a fusion of two slightly different copies of the base vector: one with a nonfunctional version of the pUC19 origin (BBa_I50020) and one with a functional version of the pUC19 origin (BBa_I50022). We corrected the synthesized BioBrick base vector (BBa_I51019) with molecular biology techniques to make the functional BioBrick base vector BBa_I51020 (see Methods). Commercial DNA synthesis processes currently rely on cloning, assembly and propagation of synthesized DNA in E. coli. Difficulties during synthesis stemmed from the inclusion of both a ccdB positive selection marker that is toxic to most E. coli strains and a synthetic origin incapable of supporting plasmid replication of the BioBrick base vector. In general, for parts whose function are incompatible with growth and replication of E. coli, the processes of DNA design and DNA synthesis cannot be easily decoupled.