Rationale: In addition to the overall abundance of undiscovered phage satellites, there are no known phage satellites that parasitize lambda. We screened for phage satellites that could infect E. coli strain EMG2 K-12, a lambda lysogen, to search for phage satellites that therefore may hijack lambda’s packaging machinery.

Procedure: We collected 4 samples of soil and water mixtures on William and Mary’s campus. Taking these samples, we made 4 enrichments of the samples and liquid culture of E. coli EMG2 K-12, an E. coli strain that is a lambda lysogen, in LB media. After 48 hours of incubation, these enrichments were filtered and plated. Two of the enrichment plates showed plaques.

Enrichment plates that show plaques and indicate potential phage satellites. Each plate shows a distinct plaque morphology.

We performed one round of plaque purification of each potential phage satellite by picking a single plaque from each enrichment plate and phage plating dilutions with E. coli EMG2 K-12. The plates from round 1 of purification were flooded with phage buffer and lysate was collected after leaving overnight for phage elution in buffer. 

Phage purification plates that were flooded to collect lysate. Purification of the potential satellite from soil sample 1 resulted in plates that did not show clear plaques. When held up to light, turbid plaques could be seen on this purification plaque. 

We then performed multiple DNA extractions to extract phage satellite DNA using the phenol:chloroform:isoamyl alcohol method (Göller et al., 2020; Spilsberg et al., 2020). DNA purities were consistently low despite repeating the chloroform:isoamyl alcohol step in an attempt to wash away phenol contamination.

DNA samples were sent to Oklahoma Medical Research Foundation for Illumina sequencing and the phage genomes were assembled using the Shovill genome assembler pipeline. 

Results: Two novel E. coli bacteriophages were discovered, a tailed phage and a filamentous phage, though these bacteriophages do not appear to be satellite phages. Both soil samples contained the filamentous phage, which shows genetic similarity with Ff filamentous phages based on its proteins. Soil sample 4 contained the novel tailed bacteriophage, which consistently lysed E. coli EMG2 K-12, as shown by clear plaques at each round of purification. 

Rationale: Traditionally, the golden rule for phage screening is to never use a lysogen to screen on; our project, however, our project developed a protocol to create lysogens to identify phage satellites. Once a phage is isolated, trying to create a stable lysogen is the second necessary step in satellite phage discovery. If a lysogen can be successfully created, we are able to proceed with screening for satellites in the future. 

Procedure: We created a bacterial lawn using M. smegmatis on top agar. Through the course of the summer, 60 lysates from William and Mary’s freshman phage lab. We performed spot test, incubated plates at 37° C for 24-48 hours, checked for mesas after incubation.

Initial spot tests of lysates. Left shows an example of a plate negative for mesas, characterized by its see through appearance where spotted. The right image shows an example of a plate positive for mesas. There is a clearing where the lysate solution was spotted with a cloudy spot within it, indicating some resistance to the phage.

If mesas were present, we streaked cells from the mesa onto a new plate and incubated at 37°C for 2-4 days. Then, we took colonies from the streaked mesa plates and performed a patch assay. After 1-4 days, we checked for halos present surrounding the patch, indicating phage release.

We streaked out positive patch assay colonies for three rounds to purify. Following purification, we inoculated colonies in liquid culture and performed a final patch assay using the same previous protocol.Finally, we tested the superinfection (homoimmunity) abilities of the putative lysogen. incubated for 24 – 48 hours at 37° and assessed the homoimmunity by plate comparison.

A good lysogen has little to no homoimmunity, such as the lysogen candidate above. The left image shows the control (lysate dilutions spotted on M. smegmatis). The right image shows the homoimmunity assay (lysate dilutions spotted on the putative lysogens). This had no plaques, showing perfect homoimmunity for this lysogen. 

Results: Out of the 55 tested lysates, five passed all of the lysogeny tests and resulted in a stable lysogen, ready to be used for satellite screening. These phages included: JollyBabz, StubbySaigey, KingChadwick, DocMcStuffins, and SucetteDeLMort in M. smegmatis. We used these to screen for satellites and tentatively identified one satellite phage in M. smegmatis.

Rationale:Mycobacterium aichiense is a strain of bacteria that conveys strong immunity to other phages due to the presence of a prophage called HerbertWM. In other words, no other known phage has been able to infect this strain of bacteria because of a temperate phage that resides in this bacteria. However, a group of high school students found a series of phage satellites that were able to infect M. aichiense and trigger the lysis of the prophage HerbertWM, killing the cell. Finding phage satellites that are able to infect a strain of bacteria that conveys phage immunity has many applications in phage therapy, and thus developing a procedure to screen for phage satellites in bacteria with prophage immunity could have many uses. 

Procedure: We collected soil from the environment and enriched it with M. aichiense. We then filtered it to get rid of the soil debris and the bacteria, leaving only media and phages. We added M. aichiense at a 1:1 ratio to the "phagelets" and then plated with top agar. After letting it grow for 24-48 hours until visible plaques, we purify plaques by taking a plug from a plaque and resuspending in phage buffer. Afterwards, we filtered and plated the full resuspended plug with M. aichiense on top agar again until webbed lysis is reached. Then the plate was flooded and we filtered the lysate and plated again with dilutions until the ideal titer was reached. 

Results: The procedure developed by our lab was given to a freshman program within the school that works with SEA-Phages to discover new phage. The students in this lab had found a putative phage satellite, but we are working on growing it to a higher titer and getting it sequenced. 

The protocol was also given to an advanced phage lab in the university, and screening for phage satellites with a prophage led a member of that lab to find a novel phage that infects M. aichiense. However, characterizing this novel phage has proved difficult thus far, and after several attempts as sequencing it seems that a large part of its genome aligns with a pathogenic bacteria called Cupriavidus gilardii, which is in not only a different species but also a different phyla of bacteria than M. aichiense. 

We also resurrected several satellites from six years ago that were thought to be degraded and lost, some of which have not been sequenced or characterized. One of those we found to be another phage satellite. The other, more interestingly, is a standard phage that bypasses homo- and hetero-immunity in M. aichiense and lyses the bacteria without excising the prophage HerbertWM. Interestingly enough, it seems to have large parts of the genome of HerbertWM but is lytic while HerbertWM is temperate. This novel phage was used in the soil microcosm as a way to compare the efficiency of phage satellites with standard phage, while the newly characterized satellite along with the other resurrected phage satellites were used to test the killing effect of M. aichiense in the soil microcosms. 

Rationale: The P4 cosmid system was originally designed by Dr. Fa-arun in University of Edinburgh (Fa-arun et al., 2023). The original purpose of this system was for CRISPR cas9 mediated cell death for disease-causing strains of bacteria such as E. coli O157:H7 and Shigella flexneri. Aspects of the P4 packaging mechanism were used to create transducing units with a CRISPR cassette. In order to create a versatile tool that could be used for both E. coli and Shigella, Dr. Fa-arun also created chimeric tail fibers to expand the host range of these transducing units. The CRISPR cas9 used in the original study targeted shiga toxins in the bacterial genomes, causing a double stranded break in the genome and thus killing the bacteria. 

In order to make the P2 lysogen package the P4 cosmid with a wide selection of tail fibers, the genes encoding for the tail fibers were removed from the genome. The P2 wild type tail fibers were compatible with the indicator strain E. coli EMG2-K12. Therefore, the plasmid with the P2 tail fibers is necessary for the packaging of the P4 cosmid. Also, the system was designed in such a way that the P2 lysogen would only package and lyse the P4 cosmid.

Procedure: First, we made chemically competent cell out of the P2 lysogen. Then, we transformed the newly competent cell with the P4 cosmid and tail fiber plasmid. Afterwards, we created a lysate by adding L-rhamnose and allowing the cells to lyse, collecting the P4 transducing units that they produce. Lastly, we quantified the transducing units by infecting the E. coli EMG2-K12 indicator strain with the lysate from the previous step

Results: During the initial trial, the lysate degraded within a week period, decreasing the titer 10-fold per day. However, after another replicate, the titer remained stable after a two week period at about 3.4*10⁹, which is higher than that of a standard phage. That supports the argument that satellite phage have a higher titer than other phage and thus a higher efficiency. 

Rationale: Satellites have the unique quality of being nonreplicative in the absence of their helper phage, providing a level of safety that standard phages do not offer. However, if there is degradation of these satellites, their inability to replicate can prevent them from having the desired effect on the system. Therefore, developing a way to maintain the high titer of the P4 transducing units can make them more efficient long-term. Since P4 requires the P2 packaging mechanism to lyse, infecting a P2 lysogen transformed with the necessary tail fibers with the P4 transducing unit and triggering lysis should produce a higher titer of transducing units

Procedure: First, we transformed electrocompetent bacteria carrying the P2 lysogen with the P2 wild type tail fiber, which has ampicillin selection. Then, we added 100uL of 10x fold diluted transducing units to 100uL of 20x concentrated bacteria. Afterwards, we incubated with shaking for 4 hours and spread 100uL on a plate with ampicillin to select for the tail fiber plasmid and chloramphenicol to select for the P4 cosmid. We then picked colonies and grew them in LB with the presence of both antibiotics, and supplement with L-rhamnose to trigger cell lysis. We collected and filtered the lysate and infected E. coli EMG2 K-12 with the lysate, diluted, and plated to quantify the transducing units.

Results: After one round of replication, there was a large increase in titer. The titer of the original transducing units was 10⁹, but after infection with only 100uL of 10x diluted transducing units, the resulting titer was 10¹¹. Therefore, there was a 100 fold increase in the titer of transducing units. 

Rationale: Before evaluating the efficacy of sequence specific killing based on a neomycin phosphotransferase gene target in the synthetic colon we needed to ensure that transducing units carrying the P4 cosmid with appropriate spacer inserts could do so in vitro. Our method for doing this is largely based on the method(s) used by Dr. Fa-Arun to characterize the original P4 cosmid.

We also wanted to ensure that any killing effect we observed was an authentic killing effect rather than one arising from a knockout of HL 713’s resistance marker while screening on kanamycin, so we adapted our procedure to account for this.

Procedure: We created transducing agent filtrate by resuspending concentrated EMG C5545 ∆cosσε ∆HG co-transformed with the Kanamycin resistance targeting P4 cosmid and a plasmid containing the wild type P2 tail fiber, in media containing L-rhamnose, which induced production of P4 transducing agents and cell lysis. We completed the procedure by filter sterilizing the lysate to produce a transducing agent filtrate free of bacteria. We verified the sterility of each filtrate by plating a portion of it on nonselective LB agar. We also created non-targeting transducing agent filtrate with the same procedure only using cells with the original P4 cosmid (i.e. lacking a targeting crRNA insert).

After producing transducing agents with the Kanamycin Resistance targeting p4 cosmid and the original P4 cosmid as a negative control, we quantified the titer of each filtrate by mixing a portion of each with a concentrated solution of the E. coli indicator strain EMG2 K-12 (ATCC 23716), performing a serial dilution of the incubated cells, and finally plating on LB plates containing chloramphenicol to screen for cosmid transductants. 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. 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.

After incubation, we serially diluted the incubated cells ten fold in LB, and plated on LB agar plates lacking antibiotics, as well as LB with antibiotics. By comparing CFU between targeting and non-targeting treatments on LB without antibiotics, Cas9 killing effect can be evaluated. Doing the same on chloramphenicol containing LB quantifies killing efficiency among transductants only.

Results: We observed a statistically significant decrease (at the 95% confidence interval) in CFU (6.26 mean fold difference) after treatment with kanamycin targeting transducing agents when compared to treatment with non-targeting transducing agents, at a multiplicity of infection of 100 - meaning estimated 100 transducing agents per HL713 cell. Screening on chloramphenicol indicated a killing efficiency among transductants of over 98% for most measurements.

Rationale: The Cas9 gene targeting system was tested in the model colon to determine the fieldability of the system and differences in killing effect in a natural environment. 

Procedure: The model colon was set up and inoculated with 100 mL HL713 in feed, made as previously described in Van den Abbeele et al. to allow the indicator strain to colonize the entire colon before inoculation with the transducing units(2010). The effluent of the model colon was regularly sampled to establish a baseline population by plating a serial dilution (10⁰, 10^-2, 10^-4, 10^-6) on LB agar plates and LB agar plates with kanamycin to screen for HL713. The model colon was inoculated with 12 mL of Cas9 targeting transducing units into the top of the first chamber, the ascending colon. 

Results: For the two model colons we inoculated with Cas9 targeting transducing units there was a statistically significant decrease in population for the effluent of model colon #1. However, while there was a decrease in the mini colon (which models one chamber of the colon), it was not statistically significant. 

Rationale: If successful, the transducing units with the RFP insert can be used as a reporter system for the presence of E. coli within the soil. When the transducing units are deployed, the infected bacteria will express a red fluorescent protein that will be visualized when bacteria plating the soil microcosms. 

Procedure: The microcosms were designed and rooted with bush bean and lettuce plants. After sufficient root growth, soil was inoculated with 18.25mLof 12.5X concentrated HL713 on August 31st. After allowing the indicator strain to colonize the soil microcosm, transducing units were inoculated. The soil microcosms were regularly sampled from the ‘center’ (where the microcosm was inoculated) and ‘edge’ (the other end of the microcosm) and plated at a 1e-4 and 1e-2 dilution respectively. The microcosms were inoculated with transducing units and sampling continued after inoculation with transducing units.

We used the Opentrons OT-2 for preparing the 96 well plate for fluorescence measurement.

Results: Ten samples were collected from distinct colonies from soil microcosm plating on LB with kanamycin and chloramphenicol. 

A1: Negative control - uninfected E. coli HL713, A2: Positive control - E. coli HL713 infected with RFP, A3: Negative control - water. A4: Sample 1, A5: Sample 2, A6: Sample 3, A7: Sample 4, A8: Sample 5, A9: Sample 6, A10: Sample 7, A11: Sample 8, A12:Sample 9. B12: Sample 10. 

The above table is a red fluorescence protein measurement for the 10 colonies from soil samples. Sample 6 showed a significantly higher measurement than the negative control suggesting that this sample was fluorescing. 

Rationale : We reasoned that we could use the same crRNA spacer insert used to create the KanR targeting P4 cosmid to characterize the silencing ability of the dCas9 P4 cosmid. If the dCas9 cosmid was working as intended HL713 transduced with the P4 cosmid should exhibit reduced resistance to kanamycin, as dCas9 would inhibit transcription elongation past its binding site, this reducing expression of neomycin phosphotransferase.

Procedure: We inserted the neomycin phosphotransferase crRNA into the dCas9 cosmid via a IIS reaction with BsaI, transformed into E. coli DH5-alpha, harvested the construct and confirmed its sequence with nanopore sequencing.

We then followed the same procedure as with previous experiments to produce transducing agent filtrate containing the dCas9 P4 cosmid (with added KanR targeting crRNA), as well as original P4 cosmid filtrate as a negative control.

Results: Treatment with dCas9 Cosmid (with added KanR targeting crRNA) transducing agent filtrate produced fewer kanamycin resistant cosmid transductants compared to a control group treated with original P4 cosmid transducing agent filtrate. Overall the experiment was not sufficiently powered to draw any definitive conclusions about the magnitude of the dCas9 cosmid’s silencing effect, but is intriguing enough to potentially motivate further characterization of this part.