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.
This vector enables multiple integrations in different positions of the same genome.
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
The default version of this vector contains the BBa_I52002 insert, 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
The DNA guide can be changed as follows:
- Be sure to have the desired guide in the RFC10 standard or a compatible one (Fig.1-a).
- Digest the guide with XbaI-SpeI (Fig.1-b).
- Digest the integrative base vector BBa_K300000 with NheI (Fig.1-c) and dephosphorylate the linearized vector to prevent re-ligation.
- 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.
- Transform the ligation in a ccdB-tolerant strain and screen the clone.
The DNA passenger can be changed as follows:
- Be sure to have the desired passenger in the RFC10 standard or a compatible one (Fig.2-a).
- Digest the passenger with EcoRI-PstI (Fig.2-b).
- Digest the integrative base vector BBa_K300000 with EcoRI-PstI (Fig.2-c).
- Ligate the digestion products (Fig.2-d).
- Transform the ligation in a pir+/pir-116 strain. Transformants with the uncut plasmid contaminant DNA do not grow because of the ccdB toxin in BBa_I52002. 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 integration in the same genome
When this vector is integrated into 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 can be eliminated each time.
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)
Materials and Methods
Plasmids and strains: the BBa_J72008 helper plasmid was kindly given by Prof. JC Anderson (UC Berkeley). MC1061 (BBa_K300078) and MG1655 (BBa_V1000) E. coli strains and the pCP20 helper plasmid were purchased from the Coli Genetic Stock Center (Yale University). DH5alpha (No part name specified with partinfo tag.) and P4 (BBa_K300978) primers had already been used in [Anderson JC et al., 2010]. The P2 (BBa_K300976) and P3 (BBa_K300977) primers have been newly designed using ApE and amplifiX. P2 and P3 have been designed also considering the previously used verification primers P2 and P3 in the pG80ko integrative plasmid, described in [DeLoache W, 2009].
Competent cells preparation: all the E. coli strains were made competent following a slightly modified version of the protocol described in [Sambrook J et al., 1989]. Briefly, cells were grown to and OD600 of ~0.4-0.6, harvested (4000 rpm, 10 min, 4°C) and the supernatant discarded. Cells were resuspended in (30 ml for each 50 ml of initial culture) pre-chilled Mg-Ca buffer (80 mM MgCl2, 20 mM CaCl2), centrifuged as before and the supernatant discarded. Cells were resuspended in (2 ml for each 50 ml of initial culture) pre-chilled Ca buffer (100 mM CaCl2, 15% glycerol), aliquoted in 0.5 ml tubes and freezed immediately at -80°C. Test the transformation efficiency as:
efficiency [CFU/ug of DNA]= # CFU * 1000 ng of DNA / amount of transformed DNA [ng]
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 169
Illegal XbaI site found at 1539
Illegal SpeI site found at 1727 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE WITH RFC[21]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2150 - 23INCOMPATIBLE 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 - 25INCOMPATIBLE 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 - 1000INCOMPATIBLE WITH RFC[1000]Plasmid lacks a prefix.
Plasmid lacks a suffix.
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