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.
 
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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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4727003 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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<partinfo>BBa_K4727002 parameters</partinfo>
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Revision as of 19:51, 24 July 2023

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.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found at 623
    Illegal suffix found at 645
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 623
    Illegal SpeI site found at 646
    Illegal PstI site found at 660
    Illegal NotI site found at 629
    Illegal NotI site found at 653
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 623
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found at 623
    Illegal suffix found at 646
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found at 623
    Plasmid lacks a suffix.
    Illegal XbaI site found at 638
    Illegal SpeI site found at 646
    Illegal PstI site found at 660
    Illegal NgoMIV site found at 127
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI.rc site found at 2026
    Illegal SapI site found at 943