Part:BBa_K300000

BioBrick integrative base vector for E. coli

BBa_K300000 is an integrative base vector backbone which can be used to integrate the desired BioBrick system into the genome of E. coli. This base vector can specialized to target the desired integration site in the host genome.

How to propagate it before performing genome integration

The default version of this vector contains BBa_I52002, so it *must* be propagated in a ccdB-tolerant strain such as DB3.1 (BBa_V1005).

After the insertion of the desired BioBrick part in the cloning site, this vector does not contain a standard replication origin anymore, so it *must* be propagated in a pir+ or pir-116 strain such as BBa_K300984 or BBa_K300985 that can replicate the R6K conditional origin (BBa_J61001).

How to perform genome integration

The integration into the E. coli chromosome can exploit the bacteriophage attP-mediated integration or the homologous recombination.

Detailed protocols about attP-mediated integration can be found here:

• Anderson JC, Dueber JE, Leguia M, Wu GC, Goler JA, Arkin AP, Keasling JD (2010), BglBricks: A flexible standard for biological part assembly. Journal of Biological Engineering 2010 4:1.
• Haldimann A, Wanner BL (2001), Conditional-replication, integration, excision, and retrieval plasmid-host systems for gene structure-function studies of bacteria. Journal of Bacteriology, 183(21), p.6386-6393.

Detailed protocols about homologous recombination can be found here:

• Martinez-Morales F, Borges AC, Martinez A, Shanmaugam KT, Ingram LO (1999), Chromosomal Integration of Heterologous DNA in Escherichia coli with Precise Removal of Markers and Replicons Used during Construction. Journal of Bacteriology, Vol. 181, No. 22, November, p. 7143–7148.
• Posfai G, Koob MD, Kirkpatrick HA, Blattner FR (1997), Versatile Insertion Plasmids for Targeted Genome Manipulations in Bacteria: Isolation, Deletion, and Rescue of the Pathogenicity Island LEE of the Escherichia coli O157:H7 Genome. Journal of Bacteriology, Vol. 179, No. 13July, p. 4426–4428.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 169
  Illegal XbaI site found at 1539
  Illegal SpeI site found at 1727
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2150
  Illegal NheI site found at 1558
  Illegal NheI site found at 1974
  Illegal SpeI site found at 2
  Illegal SpeI site found at 1727
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 2156
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2150
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 2150
  Illegal suffix found at 2
  Illegal XbaI site found at 169
  Illegal XbaI site found at 1539
  Illegal SpeI site found at 1727
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 2150
  Plasmid lacks a suffix.
  Illegal XbaI site found at 169
  Illegal XbaI site found at 1539
  Illegal XbaI site found at 2165
  Illegal SpeI site found at 2
  Illegal SpeI site found at 1727
  Illegal PstI site found at 16
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.