Difference between revisions of "Part:BBa K4201015:Design"

 
 
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===Design Notes===
 
===Design Notes===
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Notably, the backbone contains a left border overdrive sequence (nt 1-62) which greatly enhances cleavage during transformation. Moreover, the psV1 replicon The Tet resistance gene was replaced with a NPTIII kanamycin resistance gene to make transformation easier, via PCR amplification and ligation. The blue/white selection marker (nt 67-756) is flanked by BsaI cut sites on either side, allowing for blue white selection of transformed colonies.
 
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Notably, the backbone contains a left border overdrive sequence (nt 1-62) which greatly enhances cleavage during transformation<sup>4</sup>. It contains an E. coli origin of replication (nt 3584-4162) which enables plasmid replication in our model organism<sup>5</sup>. The Tet resistance gene was replaced with a NPTIII kanamycin resistance gene<sup>6</sup> to make transformation easier, via PCR amplification and ligation. The blue/white selection marker (nt 67-756) is flanked by BsaI cut sites on either side, allowing for blue white selection of transformed colonies.
  
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This backbone was designed for transformation in NEB 10-<i>beta E. coli</i> and GoldBio’s EHA105 <i>Agrobacterium</i>.
  
 
===Source===
 
===Source===
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===References===
 
===References===
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1. Lee, S. New Binary Ti Vectors with Co-directional Replicons for Agrobacterium Tumefaciens-mediated Transformation of Higher Plants. 149. <br> 2. Heeb, S. et al. Small, Stable Shuttle Vectors Based on the Minimal pVS1 Replicon for Use in Gram-Negative, Plant-Associated Bacteria. Mol. Plant-Microbe Interactions® 13, 232–237 (2000).<br>3. Yanisch-Perron, C., Vieira, J. & Messing, J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33, 103–119 (1985). <br> 4. Toro, N., Datta, A., Yanofsky, M. & Nester, E. Role of the overdrive sequence in T-DNA border cleavage in Agrobacterium. Proc. Natl. Acad. Sci. U. S. A. 85, 8558–8562 (1988).
 +
<br> 5. Hershfield, V., Boyer, H. W., Yanofsky, C., Lovett, M. A. & Helinski, D. R. Plasmid ColE1 as a Molecular Vehicle for Cloning and Amplification of DNA. Proc. Natl. Acad. Sci. U. S. A. 71, 3455–3459 (1974).
 +
<br> 6. Rosellini, D. Selectable Markers and Reporter Genes: A Well Furnished Toolbox for Plant Science and Genetic Engineering. Crit. Rev. Plant Sci. 31, 401–453 (2012).

Latest revision as of 05:32, 12 October 2022


Kan BsaI


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 354
    Illegal XbaI site found at 327
    Illegal PstI site found at 315
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 354
    Illegal NheI site found at 804
    Illegal PstI site found at 315
    Illegal NotI site found at 849
    Illegal NotI site found at 2139
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 354
    Illegal BamHI site found at 333
  • 23
    INCOMPATIBLE WITH RFC[23]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 354
    Illegal XbaI site found at 327
    Illegal PstI site found at 315
  • 25
    INCOMPATIBLE WITH RFC[25]
    Unknown
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI site found at 750
    Illegal BsaI.rc site found at 68
    Illegal SapI site found at 922


Design Notes

Notably, the backbone contains a left border overdrive sequence (nt 1-62) which greatly enhances cleavage during transformation4. It contains an E. coli origin of replication (nt 3584-4162) which enables plasmid replication in our model organism5. The Tet resistance gene was replaced with a NPTIII kanamycin resistance gene6 to make transformation easier, via PCR amplification and ligation. The blue/white selection marker (nt 67-756) is flanked by BsaI cut sites on either side, allowing for blue white selection of transformed colonies.

This backbone was designed for transformation in NEB 10-beta E. coli and GoldBio’s EHA105 Agrobacterium.

Source

This plasmid was made with a pLSUK vector. B/W selection is from pUC19 and kanamycin resistance is conveyed through a NPTIII gene.

References

1. Lee, S. New Binary Ti Vectors with Co-directional Replicons for Agrobacterium Tumefaciens-mediated Transformation of Higher Plants. 149.
2. Heeb, S. et al. Small, Stable Shuttle Vectors Based on the Minimal pVS1 Replicon for Use in Gram-Negative, Plant-Associated Bacteria. Mol. Plant-Microbe Interactions® 13, 232–237 (2000).
3. Yanisch-Perron, C., Vieira, J. & Messing, J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33, 103–119 (1985).
4. Toro, N., Datta, A., Yanofsky, M. & Nester, E. Role of the overdrive sequence in T-DNA border cleavage in Agrobacterium. Proc. Natl. Acad. Sci. U. S. A. 85, 8558–8562 (1988).
5. Hershfield, V., Boyer, H. W., Yanofsky, C., Lovett, M. A. & Helinski, D. R. Plasmid ColE1 as a Molecular Vehicle for Cloning and Amplification of DNA. Proc. Natl. Acad. Sci. U. S. A. 71, 3455–3459 (1974).
6. Rosellini, D. Selectable Markers and Reporter Genes: A Well Furnished Toolbox for Plant Science and Genetic Engineering. Crit. Rev. Plant Sci. 31, 401–453 (2012).