Part:pSB3T5:Design

Low to medium copy BioBrick standard vector

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3231
  Illegal NheI site found at 1403
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 3237
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3231
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 3231
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 3231
  Plasmid lacks a suffix.
  Illegal XbaI site found at 3246
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal AgeI site found at 994
  Illegal AgeI site found at 1317
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI site found at 289
  

Design Notes

pSB3T5 is a BioBrick standard vector. As such, it was designed to fulfill the basic demands of the biological engineers who use them. Due to issues in vector design and synthesis, it meets four of our five 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.

Source

pSB3T5 was constructed by assembling synthesized BioBrick base vector BBa_I51001, replication origin BBa_I50032 and tetracycline antibiotic resistance marker BBa_P1005 using the procedure outlined at Help:Plasmids/Construction.

References

<biblio>

1. Bernard-J-Mol-Biol-1992 pmid=1324324
2. Miki-J-Mol-Biol-1992 pmid=1316444
3. Bernard-Gene-1994 pmid=7926841
4. Bernard-Gene-1995 pmid=7557407
5. Bernard-Biotechniques-1996 pmid=8862819
6. Vieira-Gene-1982 pmid=6295879
7. Norrander-Gene-1983 pmid=6323249
8. Yanisch-Perron-Gene-1985 pmid=2985470
9. Cabello-Nature-1976 pmid=765836
10. Ioannou-Nat-Genet-1994 pmid=8136839
11. Gentz-Proc-Natl-Acad-Sci-USA-1981 pmid=6946440
12. Stueber-EMBO-J-1982 pmid=6327267
13. Bowater-Nucleic-Acids-Res-1997 pmid=9207036

</biblio>