Plasmid_Backbone

Part:BBa_K300001:Design

Designed by: Susanna Zucca, Lorenzo Pasotti, Federica Sampietro, Paolo Magni   Group: iGEM10_UNIPV-Pavia   (2010-10-03)

BioBrick integrative base vector for S. cerevisiae


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Plasmid lacks a suffix.
    Illegal XbaI site found at 46
    Illegal SpeI site found at 2
    Illegal PstI site found at 261
    Illegal PstI site found at 1607
    Illegal PstI site found at 3663
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 3727
    Illegal NheI site found at 1549
    Illegal NheI site found at 1600
    Illegal SpeI site found at 2
    Illegal PstI site found at 261
    Illegal PstI site found at 1607
    Illegal PstI site found at 3663
    Illegal NotI site found at 3733
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 3727
    Illegal BglII site found at 51
    Illegal XhoI site found at 1499
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found at 3727
    Plasmid lacks a suffix.
    Illegal XbaI site found at 46
    Illegal SpeI site found at 2
    Illegal PstI site found at 261
    Illegal PstI site found at 1607
    Illegal PstI site found at 3663
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found at 3727
    Plasmid lacks a suffix.
    Illegal XbaI site found at 46
    Illegal XbaI site found at 3742
    Illegal SpeI site found at 2
    Illegal PstI site found at 261
    Illegal PstI site found at 1607
    Illegal PstI site found at 3663
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI.rc site found at 2695


Design Notes

This vector backbone was designed as a modular integrative vector for S. cerevisiae. 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 not the same as other RFC10 compatible vectors. It contains a RFC10 BioBrick Prefix (BBa_G00000) and a SpeI restriction site instead of the original BioBrick Suffix. However, the presence of unique EcoRI and SpeI sites in the cloning site fully supports the assembly of the desired BioBrick parts in the cloning site upon EcoRI-SpeI digestion. This design feature has been forced by the presence of illegal XbaI and PstI sites in the TEF promoter in the LoxP-KanMX-LoxP cassette (BBa_K300989). This vector does not support the 3A Assembly.
  2. The two NheI sites and the two AvrII sites flanking the default integration sequences BBa_K300986 and BBa_K300987 enable the engineering of this backbone by assembling new user-defined BioBrick integration sequences upon XbaI-SpeI digestion.
  3. This vector can be propagated in E. coli at high copy thanks to the pMB1 replication origin and the Ampicillin resistance marker present in pSB1A2 (BBa_K300988), that is one of the standard parts that compose this integrative vector.
  4. Standard verification primer binding sites VF2 (BBa_G00100) and VR (BBa_G00102) are present in the pSB1A2 (BBa_K300988) backbone. They can be used to verify the vector length and sequence comprised between the two integration sites.


Genome integration features:

  1. The LoxP-KanMX-LoxP cassette (BBa_K300989) enables the selection of positive yeast integrants on YPD agar plates supplemented with 200 ug/ml of G418 geneticin. Once integrated, this cassette can be excised upon Cre recombinase activity. This allows to perform multiple (subsequent) integrations in the same strain, always using the same dominant G418 resistance marker.
  2. The heterologous modules in the LoxP-KanMX-LoxP cassette (BBa_K300989), i.e. the TEF promoter and the TEF transcriptional terminator from A. gossypii and the KanR from the Tn903 transposon of E. coli, show a very low homology with the S. cerevisiae genome. For this reason, the vector integration events in unwanted positions in the yeast genome are limited.

Source

  1. BBa_K300980, provided by Mr Gene DNA synthesis service (www.mrgene.com), was excised from its original shipping vector (pMA) through digestion with MfeI (Fermentas) and NsiI (Fermentas) restriction enzymes. It was then isolated through a 1% agarose gel electrophoresis and gel-extracted (Macherey-Nagel NucleoSpin Extract II).
  2. BBa_I763007, available in the Registry, was digested with EcoRI-PstI (Roche), run on agarose gel and its pSB1A2 vector was gel-extracted as described above.
  3. Digested BBa_K300980 and pSB1A2 have compatible ends (EcoRI-MfeI and PstI-NsiI). They were ligated with T4 Ligase (Roche) and transformed into competent TOP10 E. coli strain (BBa_V1009) which were then plated on Ampicillin (100 ug/ml) plates.
  4. Positive transformants were identified by colony PCR with VF2 (BBa_G00100) and VR (BBa_G00101) standard primers and by restriction mapping with EcoRI (Roche) or NsiI (Fermentas). The yielded plasmid had BBa_B0033 flanked by EcoRI and SpeI.
  5. The yielded plasmid was then digested with EcoRI-SpeI (Roche) and BBa_K300007 (digested with EcoRI-SpeI as well) was assembled in the vector, thus yielding BBa_K300001-BBa_K300007 (i.e. there is the BBa_K300007 part in the cloning site).

References

  1. De Antoni A, Gallwitz D (2000), A novel multi-purpose cassette for repeated integrative epitope tagging of genes in Saccharomyces cerevisiae. Gene 246, 179–185.
  2. Giaever G et al. (2002), Functional profiling of the Saccharomyces cerevisiae genome. Nature 418: 387-391.
  3. Guldener U, Heck S, Fiedler T, Beinhauer J, Hegemann JH (1996), A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Research, Vol. 24, No. 13 2519–2524.
  4. Gueldener U, Heinisch J, Koehler GJ, Voss D, Hegemann JH (2002), A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast. Nucleic Acids Res. Mar 15;30(6):e23.
  5. Kim KS, Pfeifer K, Powell L, Guarente L (1990), Internal deletions in the yeast transcriptional activator HAP1 have opposite effects at two sequence elements. Proc. Nad. Acad. Sci. USA Vol. 87, pp. 4524-4528, June, Genetics.
  6. Kim MD, Lee TH, Lim HK, Seo JH (2004), Production of antithrombotic hirudin in GAL1-disrupted Saccharomyces cerevisiae. Applied Microbiology and Biotechnology Volume 65, Number 3 / August.
  7. Mortimer RK, Johnston JR (1986), Genealogy of principal strains of the yeast genetic stock center. Genetics 113(1):35-43.
  8. Nevoigt E, Kohnke J, Fischer CR, Alper H, Stahl U, Stephanopoulos G (2006), Engineering of Promoter Replacement Cassettes for Fine-Tuning of Gene Expression in Saccharomyces cerevisiae. Applied and Environmental Microbiology, August, Vol. 72, No. 8, 5266–5273.
  9. Rohde JR, Trinh J, Sadowski I (2000), Multiple Signals Regulate GAL Transcription in Yeast. Molecular and Cellular Biology, June, p. 3880–3886 Vol. 20, No. 11.
  10. Sauer B (1987), Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae. Mol. Cell. Biol., 7, 2087–2096.
  11. Sherman F (1998), An Introduction to the Genetics and Molecular Biology of the Yeast Saccharomyces cerevisiae. University of Rochester Medical School, Rochester.
  12. Sliwa P, Korona R (2005), Loss of dispensable genes is not adaptive in yeast. PNAS, December 6, vol. 102, no.49, 17670–17674.
  13. West RW Jr, R. Rogers Yocum R, Ptashne M (1984), Saccharomyces cerevisiae GAL1-GAL1O Divergent Promoter Region: Location and Function of the Upstream Activating Sequence UASG. Molecular and Cellular Biology, November, P.2467-2478, Vol. 4. No. 11.
  14. Winzeler EA et al. (1999), Functional Characterization of the S. cerevisiae Genome by Gene Deletion and Parallel Analysis. Science 285, 901.