Difference between revisions of "Part:BBa K300000"
Line 8: Line 8:
 
The default version of this vector contains <partinfo>BBa_I52002</partinfo>, so it *must* be propagated in a ''ccdB''-tolerant strain such as DB3.1 (<partinfo>BBa_V1005</partinfo>).
 
The default version of this vector contains <partinfo>BBa_I52002</partinfo>, so it *must* be propagated in a ''ccdB''-tolerant strain such as DB3.1 (<partinfo>BBa_V1005</partinfo>).
  
−
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 <partinfo>BBa_K300984</partinfo> or <partinfo>BBa_K300985</partinfo> that can replicate the R6K conditional origin (<partinfo>BBa_J61001</partinfo>).
+
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 BW25141 (<partinfo>BBa_K300984</partinfo>) or BW23474 (<partinfo>BBa_K300985</partinfo>) that can replicate the R6K conditional origin (<partinfo>BBa_J61001</partinfo>).
 +
 
  
 
===How to engineer it===
 
===How to engineer it===
 +
  
 
===How to perform genome integration===
 
===How to perform genome integration===

Revision as of 17:59, 14 October 2010

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 BW25141 (BBa_K300984) or BW23474 (BBa_K300985) that can replicate the R6K conditional origin (BBa_J61001).


How to engineer it

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 et al., 2010 (REFERENCE 1 [1])
  • Haldimann A and Wanner BL, 2001 (REFERENCE 8 [2])

Detailed protocols about homologous recombination can be found here:

  • Martinez-Morales F et al., 1999 (REFERENCE 11 [3])
  • Posfai G et al., 1997 (REFERENCE 12 [4])


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.