BioBrick base vector, as revised by Codon Devices
This is a vector scaffold for constructing new BioBricks vectors. Information on the design is available at http://openwetware.org/wiki/Synthetic_Biology:Vectors/pSB%2A%2A5_design
Documented at http://openwetware.org/wiki/Synthetic_Biology:Vectors/pSB%2A%2A5_design Due to technical difficulties during synthesis, rather than including BBa_P1011 as designed, BBa_P1016 was synthesized as a component of this part. Hence, BBa_I51000 documents the original design and BBa_I51001 is the vector scaffold as synthesized.
This is a composite part generated from several basic parts obtained from a variety of sources.
p70-CsgA-His, His-tagged curli generator
Juliette Paillet, Alexandre Duprey, Emiel van der Kouwe
This is a targeting vector for homologous recombination into the ADE4 locus of S. cerevisiae. This vector can be used in cloning free cloning (ref. 2010 uOttawa iGEM wiki). Yeast strains with an ADE1 or ADE2 deletion or mutation have red colonies; by integrating the constructs using our protocol the ADE4 gene is knocked out and the colonies express a white phenotype.
This is a plasmid that we use for consruction and further cloning steps.
BioBrick base vector, as synthesized by Codon Devices
BBa_I51019 is the sequence of the BioBrick base vector as synthesized by Codon Devices, Inc. in Cambridge, MA. The original design of the BioBrick base vector is available as BBa_I51000. BBa_I51001 is the sequence of the BioBrick base vector as reported by Codon Devices, Inc. although the true sequence is something like to BBa_I51019. The exact sequence of the provided DNA is difficult to determine. The corrected BioBrick base vector that was successfully constructed from BBa_I51019 is BBa_I51020.
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.
BioBrick base vector (for constructing pSB**5 vectors)
Documented at http://openwetware.org/wiki/Synthetic_Biology:Vectors/pSB%2A%2A5_design Due to technical difficulties during synthesis, rather than including BBa_P1011 as designed, BBa_P1016 was synthesized as a component of this part. Hence, BBa_I51000 documents the original design and BBa_I51001 is the vector scaffold as synthesized. BBa_I52002 is the base vector as corrected from the synthesized construct.
Plasmid for Chromosomal Integration in Yeast at His3
This part is designed to allow simple chromosomal integration of BioBrick plasmids. It is based off of the bacterial pSB1AT3 backbone allowing for normal DNA manipulation to occur in bacteria. However the BioBrick cut sites are flanked by homologous regions to the His3 locus in S. cerevisiae. To transform S. cerevisiae the plasmid is cut with BbsI creating two non-sticky ends. The linear fragment then contains His3 homologous sequences at its ends that can be used to chromosomally integrate the BioBrick plasmid into the yeast genome. Transformants can then be selected for using His- medium.
This part was derived from plasmid pSB1AT3. Two successive rounds of PCR inserted a BbsI cut site and 40 bp homologous region in before the prefix and after the suffix. The following Primers were used: Forward 1 GCACAGAACAATAACCTGCTGGAAACGAAGATAAATCgaagacGATTACTTCGCGTTATGCAGGC Reverse 1 GCATCTTCTCAAATATGCTTCCCAGCCTGCTTATCcttctgAAATTCTGCCTCGTGATACGCC Forward 2 tactagtagcggccgctgcagTCTTAACCCAACTGCACAGAACAATAACCTGCTGGAAACG Reverse 2 ctctagaagcggccgcgaattcTTAGTATTGCTGGCCGCATCTTCTCAAATATGCTTCCCAGCC
BioBrick base vector, as originally designed
p70-CsgA-His*2, double His-tagged curli generator
This part is similar to [http://partsregistry.org/Part:BBa_J61002 J61002] but the backbone is based on pSB1C3 and carries a chloramphenicol resistance.
* This part is a compination of E1010, B0034 and pSB1C3 from the registry.
Plasmid for Chromosomal Integration in Yeast at Ura3
This part is designed to allow simple chromosomal integration of BioBrick plasmids. It is based off of the bacterial pSB1AT3 backbone allowing for normal DNA manipulation to occur in bacteria. However the BioBrick cut sites are flanked by homologous regions to the Ura3 locus in S. cerevisiae. To transform S. cerevisiae the plasmid is cut with BbsI creating two non-sticky ends. The linear fragment then contains Ura3 homologous sequences at its ends that can be used to chromosomally integrate the BioBrick plasmid into the yeast genome. Transformants can then be selected for using Ura- medium.