Difference between revisions of "Part:BBa K4727002"
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Revision as of 20:28, 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 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
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found at 2522
Illegal suffix found at 2544 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE 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 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found at 2522
Illegal suffix found at 2545 - 25INCOMPATIBLE 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 - 1000INCOMPATIBLE 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).