Difference between revisions of "Part:BBa K4727002"
 
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Currently, the production of engineered M13 phage particles through a helper plasmid involves constructs composed of a replication origin (ORI) that facilitates replication within E. coli, an antibiotic selection marker that allows for the selection of transformed cells, and the genes encoding the M13 phage capsid proteins. These constructs are typically obtained by removing the encapsidation sequence from the M13 phage genome and replacing it with ORI and the selection marker15. Of particular interest to us was the M13cp plasmid, built by Chasteen et al.15. However, this plasmid is not compatible with the BioBrick standard as it carries two PstI restriction sites flanking the ORI furthermore, it lacks the BioBrick prefix and suffix. By digesting the plasmid with two restriction enzymes, PstI and MluI, the region containing ORI and the selection marker (chloramphenicol resistance) could be easily removed. This region was then replaced with the standard pSB3K3 backbone, which carries the same ORI as the original helper plasmid, particularly p15A, and utilizes kanamycin resistance as the selection marker, compatible with the marker carried by the M13 phagemid pTZ19R. This allows the production of phage particles through co-transformation in a packaging cell.
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== M13 standard Helper Plasmid ==
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The production of engineered M13 phage particles through a helper plasmid involves constructs composed of a replication origin (ORI) that facilitates replication within E. coli, an antibiotic selection marker that allows for the selection of transformed cells, and the genes encoding the M13 phage capsid proteins. These constructs are typically obtained by removing the encapsidation sequence from the M13 phage genome and replacing it with ORI and the selection marker[1]. Of particular interest to us was the M13cp plasmid, built by Chasteen et al.[1]. However, this plasmid is not compatible with the BioBrick standard as it carries two PstI restriction sites flanking the ORI, furthermore, it lacks the BioBrick prefix and suffix. This region was then replaced with the standard pSB3K3 backbone, which carries the same ORI as the original helper plasmid, particularly p15A, and utilizes kanamycin resistance as the selection marker, compatible with the marker carried by the M13 phagemid <partinfo>BBa_K4727003</partinfo>. This allows the production of phage particles through co-transformation in a packaging cell.
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This part has been designed to make tropism variation easy thanks to the possibility to insert new genes for tropism determinants such as <partinfo>BBa_K4727006</partinfo> and <partinfo>BBa_K4727007</partinfo>
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<span class='h3bb'>Sequence and Features</span>
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<span class='h3bb'></span>
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==Sequence and Features==
 
<partinfo>BBa_K4727002 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4727002 SequenceAndFeatures</partinfo>
  
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==References==
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[1] Chasteen, L., Ayriss, J., Pavlik, P. & Bradbury, A. R. M. Eliminating helper phage from phage display. Nucleic Acids Res. 34, e145–e145 (2006).
  
 
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Latest revision as of 20:07, 25 September 2023

M13 standard Helper Plasmid

The production of engineered M13 phage particles through a helper plasmid involves constructs composed of a replication origin (ORI) that facilitates replication within E. coli, an antibiotic selection marker that allows for the selection of transformed cells, and the genes encoding the M13 phage capsid proteins. These constructs are typically obtained by removing the encapsidation sequence from the M13 phage genome and replacing it with ORI and the selection marker[1]. Of particular interest to us was the M13cp plasmid, built by Chasteen et al.[1]. However, this plasmid is not compatible with the BioBrick standard as it carries two PstI restriction sites flanking the ORI, furthermore, it lacks the BioBrick prefix and suffix. This region was then replaced with the standard pSB3K3 backbone, which carries the same ORI as the original helper plasmid, particularly p15A, and utilizes kanamycin resistance as the selection marker, compatible with the marker carried by the M13 phagemid BBa_K4727003. This allows the production of phage particles through co-transformation in a packaging cell.

This part has been designed to make tropism variation easy thanks to the possibility to insert new genes for tropism determinants such as BBa_K4727006 and BBa_K4727007


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found at 2522
    Illegal suffix found at 2544
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2522
    Illegal NheI site found at 1177
    Illegal SpeI site found at 2545
    Illegal PstI site found at 2559
    Illegal NotI site found at 2528
    Illegal NotI site found at 2552
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2522
    Illegal BglII site found at 2877
    Illegal XhoI site found at 813
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found at 2522
    Illegal suffix found at 2545
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found at 2522
    Plasmid lacks a suffix.
    Illegal XbaI site found at 2537
    Illegal SpeI site found at 2545
    Illegal PstI site found at 2559
    Illegal AgeI site found at 1263
    Illegal AgeI site found at 1586
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI.rc site found at 2291

References

[1] Chasteen, L., Ayriss, J., Pavlik, P. & Bradbury, A. R. M. Eliminating helper phage from phage display. Nucleic Acids Res. 34, e145–e145 (2006).