Plasmid_Backbone

Part:BBa_K300001

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

BioBrick integrative base vector for S. cerevisiae

This is an integrative vector which can be used to insert the desired RFC10-compatible BioBrick parts/devices/systems into the genome of S. cerevisiae. This vector can also be specialized to target the desired integration site in the host genome.

BioBrick integrative base vector BBa_K300001.
Parts notation.

The default version of this backbone targets the Gal system of the S288C strain (BBa_K300979) through the two homologous regions BBa_K300986 and BBa_K300987. The Gal system is not essential for yeast survival if the strain is grown on carbon sources other than galactose.

This vector enables multiple integrations in different positions of the same genome. The usage of the KanMX dominant selection marker can avoid the usage of auxotrophic markers.


Glossary

HR (Homologous Region) is a sequence that can recombine with the host genome.


How to propagate it before performing genome integration

This vector can be easily propagated in E. coli thanks to the high-copy replication origin and the Ampicillin resistance selection marker, both derived from the pSB1A2 vector backbone.


How to integrate a BioBrick into the yeast genome

  1. Digest BBa_K300001 and the desired BioBrick part with EcoRI-SpeI and ligate them (Fig.1).
  2. Propagate the resulting plasmid in E. coli and extract plasmid DNA from bacteria.
  3. Digest the resulting plasmid with SbfI to linearize the DNA of interest (Fig.2). This is known to increase the integration efficiency from 10- to 50-fold when compared to a non-linearized DNA Reference 11.
  4. Transform the linearized plasmid into S. cerevisiae and select integrants on G418 antibiotic plates (Fig.3).
Fig.1: Assembly of the desired BioBrick part into the integrative vector.
Fig.2: The plasmid can be linearized upon SbfI digestion to separate the yeast DNA fragment (containing the part of interest and the LoxP-KanMX-LoxP cassette) from the E. coli DNA fragment (containing the replication origin and the Amp resistance).
Fig.2: Integration of the BioBrick of interest into the S. cerevisiae genome by homologous recombination. In this figure, the integration in the Gal system genomic region is shown.

Users can change the integration site by engineering the vector: BBa_K300986 and BBa_K300987 are flanked by two AvrII and two NheI respectively and for this reason the two Homologous Regions can be excided. New homologous sequences compatible with RFC10 can be digested with XbaI-SpeI and assembled because AvrII, NheI, XbaI and SpeI have compatible sticky ends. Note that this assembly is not directional and the correct orientation can be validated through sequencing with standard VF2 and VR primers.

How to perform the KanMX marker excision

The KanMX dominant selection marker is flanked by two loxP recombination sites and for this reason it can be excided upon Cre recombinase activity. The Cre recombinase has to be expressed by a helper plasmid.


KanMX excision with loxP sites recombination.

Sequence and Features

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
    Illegal EcoRI site found at 3727
    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


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Parameters
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