Part:BBa_I52002:Design

ccdB and minimal pUC19 derived high copy origin

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 435
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 323
  

Design Notes

The BioBrick standard vectors are designed to 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.

BioBrick standard vectors are also designed to be easy to purify. To meet this requirement, we included a pUC19-derived origin (BBa_I50022) in addition to the ccdB selection marker within the BioBrick cloning site of BioBrick standard vectorsVieira-Gene-1982 Norrander-Gene-1983 Yanisch-Perron-Gene-1985. The high copy origin encoded by BBa_I50022 means that both base vector DNA and any derived vector DNA are easily purified in large quantities, irrespective of whether the vector replication origin is low copy or notCabello-Nature-1976 Ioannou-Nat-Genet-1994. Cloning a BioBrick part into the BioBrick cloning site removes the high copy origin in the cloning site thereby restoring replication control to the vector origin.

Source

BBa_P1016 (ccdB positive selection marker) and BBa_I50022 (minimal high copy origin derived from pUC19).

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

</biblio>

• Bernard P, Gabant P, Université Libre de Bruxelles. Cloning and/or sequencing vector US Patent Number 5,910,438, 1999. [http://www.google.com/patents?vid=USPAT5910438 Google Patents]
• Bernard P, Gabant P, Université Libre de Bruxelles. Cloning and/or sequencing vector US Patent Number 6,180,407 B1, 2001. [http://www.google.com/patents?vid=USPAT6180407 Google Patents]
• Bernard P, Gabant P, Université Libre de Bruxelles. Cloning and/or sequencing vector US Patent Number 7,176,029 B2, 2007. [http://www.google.com/patents?vid=USPAT7176029 Google Patents]