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. | |
Revision as of 17:48, 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.