Plasmid_Backbone

Part:BBa_K300000:Design

Designed by: Lorenzo Pasotti, Matteo Meroso, Nicolo' Politi   Group: iGEM10_UNIPV-Pavia   (2010-10-03)

BioBrick integrative base vector for E. coli


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.


Design Notes

This vector backbone was designed as a modular integrative vector for E. coli. Most of its features have been inspired by BBa_I51020 and BBa_J72007. In this section, the main design features for vector engineering and for the genome integration of the vector are reported.


Vector engineering features:

  1. The cloning site is compatible with the original BioBrick standard (RFC10), i.e. it is composed by the BioBrick Prefix (BBa_G00000) and Suffix (BBa_G00001). The presence of illegal restriction sites (XbaI in BBa_J72001 and SpeI in BBa_K300991) prevents the usage of this backbone in the classic BioBrick Standard Assembly process. However, the presence of unique EcoRI and PstI sites in Prefix and Suffix fully supports the assembly of the desired BioBrick parts in the cloning site upon EcoRI-PstI digestion and also supports the 3A Assembly.
  2. The two NheI restriction sites flanking the default integration guide sequence BBa_K300991 enable the engineering of this backbone by assembling new user-defined BioBrick integration guides upon XbaI-SpeI digestion, if the desired guide conforms to the RFC10 or a compatible standard.
  3. Like in many other standard vector backbones (e.g. the pSB**5 series), the binding sites for standard primers VF2 (BBa_G00100) and VR (BBa_G00102) are present upstream and downstream of the BioBrick cloning site respectively. These two sequences are sufficiently distant from the cloning site to enable a good quality sequencing of the insert.

Genome integration features:

  1. The four transcriptional terminators BBa_B0053, BBa_B0054, BBa_B0055 and BBa_B0062 ensure the transcriptional insulation of the integrated part from its flanking genome sequences.
  2. The two FRT recombination sites (BBa_J72001) enable the excision of BBa_K300994-BBa_K300998-BBa_G0001-BBa_B0025-BBa_G0001-BBa_K300999-BBa_K300995 (i.e. the R6K origin and the Chloramphenicol resistance marker) upon Flp recombinase activity. This marker excision allows users to make multiple (serial) integrations in the same strain, always using the same antibiotic resistance marker.
  3. The engineering of the integration guide allows the integration of parts in user-defined genome positions and for this reason this vector supports the integration by exploiting bacteriophage attP-mediated integration as well as homologous recombination.

Source

This section describes how this vector backbone was assembled using BioBrick parts.

  1. First step - BBa_K300982 intermediate part construction:
    1. BBa_K300983, provided by Mr Gene DNA synthesis service (www.mrgene.com), was excised from its original shipping vector (pMK-RQ) through digestion with AvrII restriction enzyme (Roche). It was then isolated through a 1% agarose gel electrophoresis and gel-extracted (Macherey-Nagel NucleoSpin Extract II).
    2. BBa_K300008 was assembled via BioBrick Standard Assembly from available parts and was excised from its vector (pSB1A2) through digestion with XbaI-SpeI (Roche). It was then isolated using the same procedure described above and it was dephosphorylated by using Antarctic Phosphatase (NEB).
    3. Digested BBa_K300983 and BBa_K300008, all having compatible sticky ends, were ligated with T4 Ligase (Roche) and transformed into competent BW23474 E. coli strain (BBa_K300985) in order to allow the ligated plasmid propagation at high copy number. This strain was necessary because the replication origin of the resulting plasmid was the conditional R6K origin (BBa_J61001).
    4. Positive transformants, grown on Chloramphenicol plates, were identified by restriction mapping with EcoRI-HindIII (Roche). The yielded plasmid was BBa_K300982.
  2. Second step - BBa_K300000 final vector construction:
    1. BBa_K300982 DNA was miniprepped, digested with EcoRI-PstI (Roche), run on agarose gel and gel-extracted as described above, in order to cut out and eliminate the RBS (BBa_B0033) between EcoRI and PstI.
    2. BBa_I763007 DNA was excised from pSB1A2 by EcoRI-PstI (Roche) digestion and isolated by gel run and gel extraction as described above.
    3. Digested BBa_K300982 and BBa_I763007 were ligated and transformed into competent BW23474 E. coli strain (BBa_K300985), selected with Chloramphenicol.
    4. Positive transformants were screened by restriction mapping with EcoRI-HindIII (Roche) and sequencing with VF2 (BBa_G00100) and VR (BBa_G00101) standard primers. The yielded plasmid was BBa_K300000 with BBa_I763007 as default insert.

All the DNA manipulations were performed according to manufacturer's protocols.

References

  1. 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 4:1.
  2. Arkin AP (2008), Setting the standard in synthetic biology. Nature Biotechnology, 26:771-774.
  3. Cherepanov PP, Wackernagel W (1995), Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene 158(1):9-14.
  4. Datsenko KA, Wanner BL (2000), One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A., Jun 6;97(12):6640-5.
  5. DeLoache W (2009), Constructing and Implementing a Transcription-Based XOR Gate in Escherichia coli through Promoter Engineering. Honors Thesis, Davidson College, Department of Biology, April 6.
  6. Diederich L, Rasmussen LJ, Messer W (1992), New cloning vectors for integration in the lambda attachment site attB of the Escherichia coli chromosome. Plasmid. Jul;28(1):14-24.
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  9. Knight T (2003), Idempotent Vector Design for Standard Assembly of Biobricks, MIT DSpace [http://web.mit.edu/synbio/release/docs/biobricks.pdf].
  10. Lutz R, Bujard H (1997), Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res. Mar 15;25(6):1203-10.
  11. 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.
  12. 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.
  13. Shetty R, Endy D, Knight T (2008), Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering, 2:5.