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 parts/devices/systems into the genome of E. coli. This base vector can be specialized to target the desired integration site in the host genome.

The default version of this backbone has the bacteriophage Phi80 attP (BBa_K300991) as integration site and an mRFP1 expression cassette (BBa_I763007) as insert.

This vector enables multiple integrations in different positions of the same genome.

BioBrick integrative base vector BBa_K300000.

Parts notation.

Glossary

The passenger is the desired DNA part to be integrated into the genome.

The guide is the DNA sequence that is used to target the passenger into a specific locus in the genome.

How to propagate it before performing genome integration

This vector 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).

NOTE: Another (early) version of this vector contains the BBa_I52002 insert, so if you have this part it *must* be propagated in a ccdB-tolerant strain such as DB3.1 (BBa_V1005). We finally decided to use the BBa_I763007 instead of BBa_I52002 because the latter was not stably propagated by the DB3.1 host strain. In fact, this plasmid occasionally gave unexpected bands on agarose gel in digestion screenings.

How to engineer it

The DNA guide can be changed as follows:

Figure 1: How to engineer the integrative base vector to assemble the desired DNA guide.

1. Be sure to have the desired guide in the RFC10 standard or a compatible one (Fig.1-a).
2. Digest the guide with XbaI-SpeI (Fig.1-b).
3. Digest the integrative base vector BBa_K300000 with NheI (Fig.1-c) and dephosphorylate the linearized vector to prevent re-ligation.
4. Ligate the digestion products (Fig.1-d). XbaI, SpeI and NheI all have compatible protruding ends. Note that the ligation is not directional, but the guide can work in both directions.
5. Transform the ligation in a pir+ or pir-116 strain and screen the clone.

The DNA passenger can be changed as follows:

Figure 2: How to engineer the integrative base vector to assemble the desired DNA passenger.

1. Be sure to have the desired passenger in the RFC10 standard or a compatible one (Fig.2-a).
2. Digest the passenger with EcoRI-PstI (Fig.2-b).
3. Digest the integrative base vector BBa_K300000 with EcoRI-PstI (Fig.2-c).
4. Ligate the digestion products (Fig.2-d).
5. Transform the ligation in a pir+/pir-116 strain and screen the clone.

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)
• Haldimann A and Wanner BL, 2001 (Reference 8)

Detailed protocols about homologous recombination can be found here:

• Martinez-Morales F et al., 1999 (Reference 11)
• Posfai G et al., 1997 (Reference 12)

How to perform multiple integrations in the same genome

When this vector is integrated in the genome, the desired passenger should be maintained into the host, as well as the Chloramphenicol resistance marker and the R6K conditional replication origin. The CmR and the R6K can be excised from the genome by exploiting the two FRT recombination sites that flank them. The Flp recombinase protein mediates this recombination event, so it has to be expressed by a helper plasmid, such as pCP20 (CGSC#7629). This enables the sequential integration of several parts using the same antibiotic resistance marker, which have to be eliminated each time before the next integration step.

Detailed protocols about homologous recombination can be found here:

• Cherepanov PP and Wackernagel W, 1995 (Reference 3)
• Datsenko KA and Wanner BL, 2000 (Reference 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.