Part:BBa_K5179101
KanR Targeting P4 Cosmid
The Kanamycin Resistance Targeting P4 Cosmid is a device which consists of a fragment of the bacteriophage satellite P4’s essential region as well as a Cas9 cassette including a crRNA which targets the neomycin phosphotransferase gene, a common screening marker which confers resistance to the antibiotic kanamycin.
Usage and Biology
Plasmids containing the KanR 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 KanR P4 cosmid in tandem with a strain of E. Coli, HL713 (AddGene bacterial strain #52931) that carries a genome integrated kanamycin resistance gene. When transduced with the KanR P4 cosmid, the Cas9 cassette induces a double stranded break of HL713’s chromosome in a site within its kanamycin resistance gene. Because E. Coli lacks a non-homologous end joining DNA repair pathway, this break causes genomic instability in HL713 and leads to cell death. We used transducing units carrying this device and HL713 to characterize the P4 cosmid systems performance at inducing sequence-specific bacterial killing in simulated soil and gut environments. In these experiments, the point of using kanamycin resistance as a target because we are specifically interested in killing bacteria with this gene, but rather it works as a useful proxy for an arbitrary toxin gene, virulence factor, or other target gene of clinical or scientific interest.
We created KanR targeting P4 cosmid transducing units 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 4825
Illegal NotI site found at 662 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1185
Illegal BamHI site found at 7104
Illegal BamHI site found at 8102
Illegal XhoI site found at 2867 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 3095
Illegal SapI.rc site found at 8031
Characterization
William-and-Mary 2024
Quantification of KanR Targeting P4 Cosmid Killing Effect on E. coli HL713
After producing transducing agents containing the Kanamycin Resistance targeting P4 cosmid and containing the original P4 cosmid lacking a targeting crRNA as a negative control, we measured the titer of each on an E. Coli indicator strain lacking kanamycin resistance. We used these data to equalize the titer of each transducing agent filtrate we produced for these experiments to 10^9 transducing agents per milliliter. We measured the killing effect of each type of transducing agent filtrate by incubating 100uL of dilute transducing agent filtrate with 100uL HL713 bacterial culture diluted in SM buffer to a concentration of 10^7 cells per milliliter. Thus, all experiments were conducted at a multiplicity of infection of 100 transducing agents per target cell. HL 713 was grown up to the end of log phase as indicated by an OD600 of between 0.5-0.6 before being diluted to the desired concentration.
To determine absolute killing effect post-treatment cells were serially diluted in LB and plated on nonselective LB agar, then incubated at 37 C for 16 hours. Colonies were then counted at each dilution level to estimate post treatment bacterial titer.
|
Date of Measurement (DD/MM/YYYY) |
Targeting CFU |
Non-Targeting CFU |
Fold difference CFU |
|
31/07/2024 |
6.66x10^4 |
7x10^5 |
10.5 |
|
30/07/2024 |
1.66x10^4 |
1.66x10^6 |
100 |
|
29/07/2024 |
1x10^5 |
3.33x10^5 |
3.33 |
|
26/07/2024 |
5.67x10^5 |
2x10^6 |
3.53 |
A two sample t-test was used with the alternative hypothesis that the true difference in means between KanR transducing agent treated and control groups is less than zero. Our experimental data was sufficient to reject the null hypothesis at a significance level of 0.04 with an effect size of 6.26 fold difference between post treatment colony forming units of each group.
Quantification of Killing Efficiency
By repeating the same procedure used to quantify KanR cosmid killing effect on HL 713 and plating on chloramphenicol containing LB we can screen for cosmid transductants only. Since the same titer of transducing agents and target bacteria was used between measurements, a similar number of transductants should be generated in each round of measurement, thus any differences in the number of chloramphenicol resistant colonies between KanR targeting and non-targeting treatments arises from the killing effect of Cas9 mediated cleavage induced by the KanR targeting cosmid.
|
Date of Measurement (DD/MM/YYYY) |
Targeting CFU on Chloramphenicol |
Non-Targeting CFU on Chloramphenicol |
Killing Efficiency among Transductants |
|
18/07/2024 |
2.33x10^6 |
4.33x10^8 |
0.99 |
|
17/07/2024 |
6.66x10^6 |
3.33x10^8 |
0.98 |
|
8/07/2024 |
1.66x10^4 |
3.33x10^4 |
0.50 |
Analysis of data from three trials conducted in this way showed a killing efficiency between 0.50 and 0.99. Meaning in cases of successful transduction the KanR targeting cosmid kills its host between 50% to 99% percent of the time. Cases of HL713 colonies surviving transduction with the KanR targeting P4 cosmid could be due to a variety of factors, including the possibility of HL713 escaping the effects of sequence specific killing through a mutation to the protospacer adjacent motif in its kanamycin resistance marker, which would effectively prevent Cas9 from cleaving HL713’s chromosome.
We chose the target site specified by the crRNA spacer in the KanR targeting cosmid in specifically so that silent mutations (mutations which do not change how a protein is translated) in the relevant protospacer adjacent motif were impossible, thus immunity to the sequence specific killing effect of the KanR targeting P4 cosmid and full kanamycin resistance should be mutually exclusive. Additional analysis of the same measurements indicate this design strategy may have been successful, as when screened for cosmid transductants which have functional kanamycin resistance genes (by screening on LB containing both kanamycin and chloramphenicol) the killing efficiency recovers to high levels (96-99%).
|
Date of Measurement (DD/MM/YYYY) |
Targeting CFU on Chloramphenicol + Kanamycin |
Non-Targeting CFU on Chloramphenicol + Kanamycin |
Killing Efficiency among Transductants |
|
18/07/2024 |
3.33x10^6 |
4.67x10^8 |
0.99 |
|
17/07/2024 |
7.0x10^5 |
5.0x10^8 |
0.99 |
|
8/07/2024 |
6.66x10^3 |
1.66x10^5 |
0.96 |
| None |