Part:BBa_K5179110
BioBrick Compatible P4 Cosmid
The BioBrick Compatible P4 Cosmid is a minimal version of the P4 cosmid (Fa-Arun et al., 2023) generalized for compatibility with standardized biological parts.
Usage
Plasmids under 11.6kb using the BioBrick Compatible P4 Cosmid as its backbone can package into the capsids of phage P2, just as the bacteriophage satellite P4 can. In the presence of P2 late genes, P2 terminase will create staggered nicks in the cos site on the device, linearizing any plasmid the device is on, and package it inside a P2 capsid. The backbone also includes the P4 sid operon which codes for proteins which interface with P2 capsid proteins and cause them to take on an alternative geometry with reduced volume. This effectively excludes large DNA molecules (>30kb) from being packed, such as copies of P2’s chromosome, but permits smaller molecules around the size of P4’s genome (~11.6kb) to pack. P2 structural genes will assemble with the packaged capsids to form mature transducing agents, which can then be used to transduce a plasmid using this backbone into new bacterial hosts.
P4 cosmid transducing units can be created by co-transforming a plasmid carrying the device along with a plasmid expressing the P2 tail fiber protein into E. Coli EMG C5545 ∆cosσε ∆HG (AddGene Bacterial strain #197132), which is lysogenic for a mutant P2 phage which lacks its cos site and tail fiber protein, and has been engineered so that expression of its late genes are expressed by an L-Rhamnose promoter. This bacterial strain, engineered by Dr. Fa-Arun (Fa-Arun et al., 2023), was specially designed for use with the P4 cosmid system. We have shown it to work well with our iterations on the original P4 cosmid and we recommend using it when producing transducing units with this part.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 6104
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NotI site found at 9
Illegal NotI site found at 4100
Illegal NotI site found at 6110 - 21INCOMPATIBLE WITH RFC[21]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 6104
Illegal BamHI site found at 4623 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found at 6104
Illegal suffix found at 2 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found at 6104
Plasmid lacks a suffix.
Illegal XbaI site found at 6119
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NgoMIV site found at 1659
Illegal AgeI site found at 2901 - 1000INCOMPATIBLE WITH RFC[1000]Plasmid lacks a prefix.
Plasmid lacks a suffix.
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