Part:BBa_I51001:Design requirements

1. BioBrick standard vectors must be able to propagate any BioBrick standard biological part. To meet this requirement, the base vector must contain a complete BioBrick cloning site as specified in the original technical standardKnight-2003.
2. BioBrick standard vectors must be easy to use for their most common purpose: assembly of BioBrick standard biological parts. To meet this requirement, we included BBa_P1016 within the BioBrick cloning site. BBa_P1016 encodes the positive selection marker ccdB. Positive selection markers prevent one of the most common problems during assembly of BioBrick parts: contamination of the ligation reaction with uncut plasmid DNASambrook-2001. Any cells transformed with the uncut plasmid DNA produce the lethal protein ccdB and dieBernard-Gene-1994 Bernard-Gene-1995 Bernard-Biotechniques-1996. Note, however, that the drawback of this solution is that inclusion of this part requires that users propagate both the base vector and any derived vectors in E. coli strains tolerant of ccdB expression such as DB3.1Bernard-J-Mol-Biol-1992 Miki-J-Mol-Biol-1992.
3. BioBrick standard vectors must stably propagate BioBrick parts. Previous work suggests that transcriptional isolation of the cloned DNA fragment can make those fragments more amenable to manipulationGentz-Proc-Natl-Acad-Sci-USA-1981 Stueber-EMBO-J-1982 Bowater-Nucleic-Acids-Res-1997 Godiska-2005. Thus, to meet this requirement, we included transcriptional terminators in both directions flanking the BioBrick cloning site (BBa_B0053, BBa_B0054, BBa_B0055 and BBa_B0062). By doing so, we sought to insulate the proper maintenance and propagation of the vector from the possibly disruptive function encoded by the BioBrick part cloned into the vector. We also flanked the BioBrick cloning site on both sides with translational stop codons in all six reading frames (BBa_B0042) to ensure translational insulation as well.
4. BioBrick standard vectors should allow users to verify the length and sequence of the inserted BioBrick part. To meet this requirement and to support backwards compatibility with existing BioBrick parts and vectors, we incorporated the same primer annealing sites found in existing BioBrick vectors like pSB1A3-P1010 (BBa_G00100 and BBa_G00102). BBa_G00100 and BBa_G00102 are sufficiently distant from the BioBrick cloning site to ensure good quality sequence reads of any inserted part.

We had an additional design requirement that BioBrick standard vectors should be easy to purify. To meet this requirement, our original design included a pUC19-derived origin (BBa_I50020) in addition to the ccdB selection marker within the BioBrick cloning siteVieira-Gene-1982 Norrander-Gene-1983 Yanisch-Perron-Gene-1985. However, mutations introduced into BBa_I50020 rendered the part non-functional as a replication origin. Thus, BioBrick standard vectors with BBa_I51001 as the default cloned part fail to meet this design requirement. Nevertheless, BioBrick standard vectors with BBa_I51001 as the default cloned part are still functional.