Part:BBa_K5179105
RFP P4 Cosmid
The RFP P4 cosmid is an iteration on the P4 cosmid. The original P4 cosmid, designed by Dr. Fa-Arun (Fa-Arun et al., 2023), carried a Cas9 cassette to induce sequence specific killing by inducing a double stranded break in a gene target, the RFP cosmid was created by replacing the Cas9 cassette with an mRFP reporter device.
Usage and Biology
Plasmids containing the RFP cosmid 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 device 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 containing this device into new bacterial hosts.
In our project we used the RFP cosmid cosmid to create transducing units which visibly reported successful transduction. We did this 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]Illegal NheI site found at 3406
Illegal NheI site found at 3429
Illegal NotI site found at 662 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1185
Illegal XhoI site found at 2867 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 4021
Illegal AgeI site found at 4133 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 3095
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