Difference between revisions of "Part:BBa K5143005"
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     <h1>Description</h1>
 
     <h1>Description</h1>
 
     <p>
 
     <p>
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       pUC57 backbone we used is composed of the Ampicillin Resistance gene (to select transformant colonies), the replication origin ColE1 (enables the plasmid replication in <i> E. coli </i> in high-copy-number), the URA3 selection marker (enables the selection of recombinant yeast) and the Ura3' and Ura5'homologies region (enables the recombination of gene of interest in the yeast).
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       This backbone plasmid was linearized by PCR and used to clone various genetic elements with a view to integrating them into the V chromosome of Saccharomyces cerevisiae BY4741 yeast, which lacks the ura3 gene. To do this, it has homology sequences at the locus of interest (accession number GSE94851 on SGD), so that the recombination cassette can be integrated there (release of the cassette of interest by XhoI digestion of the plasmid). <br>
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<br>
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In addition, this backbone plasmid possesses a high-copy-number colE1 origin of replication, a selection marker for Beta-lactamase production conferring ampicillin resistance (to select colonies following transformation into E. coli DHA alpha), an auxotrophy marker ura3 (to select yeasts that have integrated the fragment of interest) and the homogeneity zones described above. <br>
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<br>
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It was used to create 3 plasmids: <a href="https://parts.igem.org/Part:BBa_K5143025">plasmid D</a>, <a href="https://parts.igem.org/Part:BBa_K5143027">plasmid venus/ruby</a> and <a href="https://parts.igem.org/Part:BBa_K5143028">plasmid Bt-toxin</a>. <br>
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     </p>
 
     </p>
 
     <img src="https://static.igem.wiki/teams/5143/backbone-plasmid-bba-k5143005.png" width="400" alt="Backbone">
 
     <img src="https://static.igem.wiki/teams/5143/backbone-plasmid-bba-k5143005.png" width="400" alt="Backbone">

Revision as of 16:14, 27 September 2024

Protein Description

Description

This backbone plasmid was linearized by PCR and used to clone various genetic elements with a view to integrating them into the V chromosome of Saccharomyces cerevisiae BY4741 yeast, which lacks the ura3 gene. To do this, it has homology sequences at the locus of interest (accession number GSE94851 on SGD), so that the recombination cassette can be integrated there (release of the cassette of interest by XhoI digestion of the plasmid).

In addition, this backbone plasmid possesses a high-copy-number colE1 origin of replication, a selection marker for Beta-lactamase production conferring ampicillin resistance (to select colonies following transformation into E. coli DHA alpha), an auxotrophy marker ura3 (to select yeasts that have integrated the fragment of interest) and the homogeneity zones described above.

It was used to create 3 plasmids: plasmid D, plasmid venus/ruby and plasmid Bt-toxin.

Backbone

Construction

This part has been synthesized in order to have the URA3 gene and the Ura3'/Ura5' homologies region.

References

mettre des réf



Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Plasmid lacks a prefix.
    Illegal EcoRI site found at 3440
    Illegal EcoRI site found at 3967
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 3440
    Illegal EcoRI site found at 3967
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 3973
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 3440
    Illegal EcoRI site found at 3967
    Illegal XhoI site found at 1605
    Illegal XhoI site found at 3446
  • 23
    INCOMPATIBLE WITH RFC[23]
    Plasmid lacks a prefix.
    Illegal suffix found at 2
    Illegal EcoRI site found at 3440
    Illegal EcoRI site found at 3967
  • 25
    INCOMPATIBLE WITH RFC[25]
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 3440
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal SapI.rc site found at 714