Overview

CAU-China is committed to establishing a highly controllable, secure, and efficient precision antibacterial platform for the production of bacteriophage particles. The purpose of this page is to present the results of the design to provide experimental evidence for the validation of the ideas.

1. Verification of Phage-like particle Synthesis and Infectivity

To enhance visualization during the verification of Phage-like particle synthesis and infectivity, we replaced the MazF toxin gene on the plasmid with bjGFP and conducted the following experiments.

Figure 1: The diagram of packaging visualization plasmid.

Plasmid construction and validation

Using our company’ s synthetic pUC19-T7 packaging sequence plasmid as a backbone, we linearized the vector via reverse PCR and then performed seamless cloning with the PTetA and bjGFP containing homologous arms. The recombinant plasmid was chemically transformed into competent Escherichia coli MG1655 cells and plated on the LB solid medium containing ampicillin. After overnight incubation at 37°C, colonies were preliminarily screened for the recombinant plasmid via colony PCR. The correct colonies were inoculated into Ampicillin-resistant LB liquid medium under shaking condition overnight, and the plasmids were extracted and then subjected to PCR using the universal primer M13F/R (Figure 2).

Sequencing results indicate that all three plasmids were successfully constructed. T1 and T2 contained minor mutations, while T3 was error-free. T3 was used for subsequent experiments.

Figure 2: The results of plasmid PCR. The expected band size should be 2028 bp, and all three detected bands yield results consistent with expectations.
M：Star Marker D5000 Ladder; 1-3: Validation Results for the pUC19-T7 Packaging Sequence-TetA-bjGFP Construct, Designated as T1, T2, and T3

Verification of Correct Phage-Like Particle Synthesis

Escherichia coli MG1655 containing the pUC19-packaging-PTetA-bjGFP plasmid (T3) was co-cultured with WT T7 until most bacteria lysed. Add 0.2 mL chloroform, vortex vigorously, centrifuge, and collect the supernatant. Filter through a 0.22 μm membrane to obtain T7 Mix (a mixture of phage-like particles and residual components). T7 Mix was thoroughly treated with RNase and DNase to digest any residual plasmid DNA and other impurities, yielding T7 Mix 2.0. We subjected T7 Mix 2.0 to a heat lysis reaction to release DNA from the capsid head of the phage-like particles. Universal primers M13-F/R were then used to detect specific bands via PCR.

Following PCR, agarose gel electrophoresis revealed the target specific band, and sequencing confirmed the sequence was entirely correct. Simultaneously, PCR performed on the control WT T7 lysate did not yield any relevant specific bands, ruling out background interference (Figure 3).

Figure 3: The results of PCR performed on T7 Mix 2.0 after thermal cracking.
M：Star Marker D5000 ladder; 1: The PCR products from the T7 Mix 2.0 heat-lysed samples; 2: The PCR products from the WT T7 phage heat-lysed samples

Verification of Phage-Like Particle Infectivity

We cultured WT Escherichia coli MG1655 in liquid LB medium and added the phage inhibitor (CRGEN phage inhibitor, PR0401) at a 1:500 ratio. When the optical density reached 0.7, WT Escherichia coli MG1655 was mixed with T7 Mix 2.0 and cultured in liquid LB for 1.5 hours. The cells were then harvested, resuspended three times in LB to wash off T7 Mix as thoroughly as possible. Finally, the cells were spread onto LB plates containing 50 μg/mL ampicillin (Amp) and incubated at 37°C overnight.

We observed target colonies emitting green fluorescence, confirming that the phage-like particles successfully infected their host bacteria and injected the DNA concatemer into WT Escherichia coli MG1655. The reporter gene bjGFP was successfully expressed.

Figure 4: Phage inhibitor-treated E. coli MG1655 co-cultured with T7 Mix 2.0.

To further validate the successful expression of bjGFP on the DNA concatemer in the recipient bacteria, we picked fluorescent colonies from Figure 4 and cultured them overnight in ampicillin-resistant LB liquid medium as the experimental group. Concurrently, WT Escherichia coli MG1655 was cultured overnight in LB liquid medium as the control group.

Results showed that the overnight culture of the experimental group exhibited intense green fluorescence upon UV excitation. In contrast, the control group showed no fluorescent signal under identical conditions (Figure 5). After centrifuging both groups to collect bacterial cells, this difference remained clearly visible under natural light. The bacterial pellet from the experimental group exhibited a characteristic green color due to bjGFP expression, while the control group’ s pellet appeared as the typical milky white color. This phenomenon strongly demonstrates the stable and high-level expression of the bjGFP gene on the DNA concatemer delivered by phage-like particles within the recipient bacteria (Figure 6).

Figure 5. Visible color difference of overnight culture between the control and experimental groups.
Left: overnight culture of the experimental group. Right: overnight culture of the control group.

Figure 6. Visible color difference of cell pellets between the control and experimental groups.
Left: overnight culture of the experimental group. Right: overnight culture of the control group.

2. Functional Validation of Toxin-Antitoxin System

Due to time constraints, we have not yet integrated the designed MazE-carrying fragment into our chassis organism genome. Therefore, we opted to construct the following toxin-antitoxin (TA) system for preliminary functional validation.

Construction and Validation of the Four Plasmids

Using pUC19 as the backbone, we linearized the vector via reverse PCR. Subsequently, we obtained the araC-pBAD DNA fragment from pKD46 and the DNA fragments encoding the antitoxin gene MazE and toxin gene MazF from the WT Escherichia coli MG1655 genome. These were then seamlessly cloned. Subsequently, the recombinant plasmids were chemically transformed into DH5α and spread onto Amp-resistant LB solid medium. After overnight incubation at 37°C, colonies were preliminarily screened for recombinant plasmids via colony PCR. The correct colonies were inoculated into ampicillin-resistant LB liquid medium under shaking incubation overnight. Plasmids were extracted and subjected to PCR verification.

Sequencing results confirmed successful construction of all four plasmids. We selected M1, M2, M3-1, and M4-1 for subsequent functional validation of the TA system.

Figure 7: Four diagrams used to validate the toxin-antitoxin system.

Figure 8: The results of plasmid PCR. The correct band using M1 as the template should be 1553bp. The correct band using M2 as the template should be 1807bp.
M: Star Marker D5000 ladder; 1: The PCR results for M1 using jc1-F and jc2-R primers; 2: The PCR results for M2 using jc1-F and jc2-R primers

Figure 9: The results of plasmid PCR. The correct band using M3 as the template should be 1922bp. The expected band using M4 as the template should be 2185bp.
M: Star Marker D5000 ladder; 1: The PCR results for M3-1 using jc1-F and jc2-R primers; 2: The PCR results for M3-2 using jc1-F and jc2-R primers; 3: The PCR results for M4-1 using jc1-F and jc2-R primers; 4: The PCR results for M4-2 using jc1-F and jc2-R primers

Functional Validation of the TA system in E. coli MG1655

We individually transformed the four successfully constructed plasmids into our chassis strain, E. coli MG1655, and tested cell toxicity on plates with or without 0.2% L-arabinose. When MazE or MazE-MazF were induced by 0.2% L-arabinose, transformant colonies were formed on the plate. However, when MazF was induced by 0.2% L-arabinose in the presence of 0.2% arabinose, no colonies were formed (Figure 10).

We also examined the effects of each group of transformed bacteria on inducing cell growth in liquid culture. As shown in Figure 11, growth inhibition was immediately observed in the MazF group following induction starting at 90 min, upon the presence of 0.2% arabinose, whereas no such phenomenon was observed in the MazE group or the MazE-MazF group. The pBAD group served as the negative control. The results indicate that MazF, as the toxin in the TA system, significantly inhibits cell growth, while MazE, as the antitoxin within the same operon, effectively neutralizes the toxicity of MazF.

Figure 10: Toxicity of MazF on plates.

Figure 11: Effect of MazF on cell growth induction.

Functional Validation of the TA system in Pseudomonas syringae pv. tomato DC3000

We individually transformed the four successfully constructed plasmids into Pseudomonas syringae pv. tomato DC3000, and cell toxicity was tested on plates with or without 0.2% L-arabinose. When MazE or MazE-MazF were induced, transformant colonies were formed on the plate. However, when MazF was induced in the presence of 0.2% arabinose, no colonies were formed (Figure 12).

Figure 12: Toxicity of MazF on plates.

In our TA system, MazF effectively inhibits the growth of the chassis strain E. coli MG1655 and the pathogenic strain DC3000 upon induction, leading to their death. The complementary MazE antitoxin effectively antagonizes MazF toxicity, ensuring the normal survival of the chassis strain. This TA system provides crucial safety assurance for our subsequent work in safely producing phage-like particles carrying toxin genes within the chassis strain.

Split the T7 genome and construct an inducible expression system for controlled production of phage-like particles

To enable E. coli MG1655 to autonomously produce the phage protein capsid and ensure that plasmids containing replication origins can undergo rolling-circle replication in E. coli MG1655 in the same manner as the T7 genome, we performed segmentally recombining the T7 genome (with gp1 removed) into the genome of E. coli MG1655 using pKD46-SacB (carrying the SacB gene for sucrose sensitivity screening).

Construction and Verification of Phage Genome Recombinant Vector

To achieve genetic recombination, we constructed four plasmids inserted with four segments of the phage genome. Using homologous double crossover, we accomplished the integration of the T7 phage genome into the genome of MG1655.

In order to ensure the integrity of the T7 phage structural proteins during this part of the validation, we maintained the integrity of the structural protein region-Class III genes by not deleting gp17 in our experiments.

Figure 13: Four diagrams of T7 phage genome recombination

We extracted phage genomic DNA from the supernatant of the lysate using the λ Phage Genomic DNA Rapid Extraction Kit, then diluted it to a concentration of 10 ng/μL for PCR amplification of genomic fragments, and successfully obtained all target genomic fragments (Figure 13).

Figure 14: The result of genome PCR. 1-1: 2910bp; 1-2: 1760bp; 2: 11438bp; 3: 9289bp; 4-1: 12421bp; 4-2: 434bp. The size of each band was correct and the sequencing results proved the correctness of the fragments.
M: Star Marker 1kb Ladder; 1: Template amplification obtained by thermal lysis 4-2; 2-7: Extraction of phage genome as template amplification of fragments T7g-1-1, T7g-1-2, T7g-2, T7g-3, T7g-4-1, T7g-4-2

To achieve the screening of homologous recombination via double crossover, we constructed the pKD46-SacB vector by inserting the promoter and coding sequence of SacB into the pKD46 vector.

Figure 15: The result of pKD46-SacB test. The correct band should be 2282 bp, and all nine band results are correct.
M: Star Marker D5000 Ladder; 1-9: pKD46-SacB test

During the construction of the four recombinant plasmids, the homologous arms for seamless cloning carried by Down-armT7 are different. Therefore, we amplified three types of Down-armT7 with different homologous arms from the genome of E. coli MG1655 using PCR, and simultaneously amplified Up-armT7 with homologous arms.

We constructed the backbone of the first recombinant plasmid, pKD46-SacB-up-down, by performing seamless cloning of pKD46-SacB linearized via inverse PCR with Up-armT7 and Down-armT7-1. For the backbones of the second and third recombinant plasmids, we conducted seamless cloning of linearized pKD46-SacB with Down-armT7-2. The backbone of the fourth recombinant plasmid was constructed by seamless cloning of linearized pKD46-SacB with T7g-4-2 and Down-armT7-3. The above products were individually transformed into E. coli DH5α via the heat shock method, plated on LB agar plates containing Amp, and incubated at 28°C. The construction of the plasmids was verified by colony PCR.

Figure 16: The result of pKD46-SacB-up-down test. The correct band should be 1168 bp, and all eight band results are correct.
M: Star Marker D5000 Ladder; 1-8: pKD46-SacB-up-down test

Figure 17: The result of pKD46-SacB-down test. The correct band should be 666 bp, and all three band results are correct.
M: Star Marker D5000 Ladder; 1-3: pKD46-SacB-down test

Figure 18: The result of pKD46-SacB-4-2-down test. The correct band should be 1076 bp, and all two band results are correct.
M: Star Marker D5000 Ladder; 1-2: pKD46-SacB-4-2-down test

After obtaining the required vectors, we extracted the corresponding three plasmids from E. coli DH5α and linearized them via inverse PCR. We constructed pKD46-SacB-1-1 by performing seamless cloning of the linearized pKD46-SacB-up-down with T7g-1-1. For the linearized pKD46-SacB-down, we conducted seamless cloning with T7g-2 and T7g-3 respectively to construct two recombinant plasmids: pKD46-SacB-2 and pKD46-SacB-3. Additionally, we constructed the recombinant plasmid pKD46-SacB-4 by seamless cloning of the linearized pKD46-SacB-4-2-down with 4-1. The above seamless cloning products were transformed into E. coli DH5α via the heat shock method, plated on LB agar plates containing Amp, and incubated at 28°C. The construction of the plasmids was verified by colony PCR.

Figure 19: The result of pKD46-SacB-1-1 test. The correct band should be 4032 bp, and 1、2、4 band results are correct.
M: Star Marker 1kb Ladder; 1-4: pKD46-SacB-1-1 test

Figure 20: The result of pKD46-SacB-2 test and pKD46-SacB-3. The correct band should be 483 bp(pKD46-SacB-2) and 503 bp(pKD46-SacB-3), and all five band results are correct.
M: Star Marker 1kb Ladder; 1-2: pKD46-SacB-2 test; 3-5: pKD46-SacB-3 test

Figure 21: The result of pKD46-SacB-4 test. The correct band should be 400 bp, and 1 and 3 band results are correct.
M: Star Marker D5000 Ladder; 1-3: pKD46-SacB-4 test

After obtaining the required vectors, we extracted the corresponding three plasmids from E. coli DH5α. We linearized pKD46-SacB-1-1 via inverse PCR, and then performed seamless cloning of this linearized vector with T7g-1-2 to construct pKD46-SacB-1. The above seamless cloning product was transformed into E. coli DH5α by the heat shock method, plated on LB agar plates containing 50 μg/mL ampicillin (Amp), and incubated at 28°C. The construction of the plasmid was verified by colony PCR.

Figure 22: The result of pKD46-SacB-1 test. The correct band should be 260 bp, and 1、2、3 band results are correct.
M: Star Marker D5000 Ladder; 1-5: pKD46-SacB-1 test

Expression Verification of Segmented Phage Genomes on the Recombinant Vector

We extracted the plasmids (pKD46-SacB-1, pKD46-SacB-2, pKD46-SacB-3, and pKD46-SacB-4) respectively from four E. coli DH5α strains transformed with the recombinant plasmids. To verify whether the genomic fragments inserted into pKD46-SacB can be normally expressed, we transformed the recombinant plasmids into E. coli BL21 strain. With the help of T7 RNA polymerase expressed by this strain under IPTG induction, we provided conditions for the expression of the fragment genes.

We separately transformed the four plasmids into four aliquots of E. coli BL21 competent cells, then plated the transformed cells on Amp-containing agar plates. After screening, we inoculated the four E. coli BL21 strains harboring the recombinant plasmids into cultures supplemented with IPTG, followed by shaking incubation. When the bacterial culture reached an OD600 value of 1.0, the bacterial cells were collected by centrifugation. For each sample, 1 mL of bacterial cells was mixed with 80 μL of PBS and 20 μL of 5× loading buffer; after boiling water bath treatment, whole bacterial protein samples were prepared. SDS-PAGE was performed on the whole bacterial proteins (Figure 22).

Figure 23: The result of bacterial whole protein SDS-PAGE Coomassie Brilliant Blue Staining.
M: Color Prestained Protein Marker; 1: WT BL21; 2: BL21 with pKD46-SacB; 3: BL21 with pKD46-SacB-1; 4: BL21 with pKD46-SacB-2; 5: BL21 with pKD46-SacB-3; 6: BL21 with pKD46-SacB-4.

From the result figure, a comparison between Lane 1 and the other lanes shows that the total bacterial proteins of E. coli BL21 containing the plasmid have an additional protein band near the 75 kDa position (the position indicated by the arrow in the figure 22). This indicates that after the pKD46-SacB vector entered the bacteria, it expressed proteins from the plasmid, altering the protein composition of the bacteria. When comparing Lanes 2 to 6, the genomic fragments inserted into the plasmid had no detectable effect on the bacterial proteins. There was no difference in the total protein amount of bacteria containing pKD46-SacB with inserted genomic fragments compared to those containing pKD46-SacB alone. It is inferred that the expression level of proteins encoded by the genomic fragments is very low. Since no additional bands (compared to Lane 2) were observed in Lanes 3–6, it is inferred that the expression level of proteins encoded by the genomic fragments is too low to be detected by SDS-PAGE.

Due to time constraints, we were unable to fully verify whether the gene fragments could express proteins normally. In fact, the segmentation of the genome and the expression of T7 RNA polymerase may both be factors contributing to the undetectable expression level of proteins encoded by the genomic fragments.

First, the phage genome contains numerous protein sequences with positional functions. When the genome was segmented into 4 plasmids for verifying protein expression, this segmentation might have blocked the interaction between proteins that are far apart on the genome, thereby affecting protein expression.

Second, the protein amounts of total bacterial proteins and phage proteins are not on the same order of magnitude. When the total bacterial proteins are clearly visible, the content of proteins expressed from the phage genome may be extremely low, making it impossible to distinguish and identify them.

Furthermore, since the genomic fragments were directly amplified from wild-type T7 without any optimization or modification, the coding regions are not suitable for expression under the specific expression conditions we established.

To achieve our goal, we may need to further refine and modify the phage genome, and appropriately optimize the sequences of the genomic fragments, so that they can be adapted to expression in the E. coli genome.

Functional Verification of Tail Fiber-Replaced PLPs

Tail Fiber Replacement

1.1 Verification of Homologous Recombination

Using the commercially synthesized pUC19-VO98_215 plasmid as a backbone, we obtained the vector-homologous recombinant fragment via nested PCR. The pUC19 vector was linearized via reverse PCR and seamlessly cloned with the recombinant fragment. The recombinant plasmid was transformed into competent Escherichia coli MG1655 cells using heat shock transformation, plated on the Amp-resistant LB solid medium, and incubated overnight at 37°C. Preliminary screening for strains harboring the recombinant plasmid was performed via colony PCR and colony PCR was performed in triplicate, and all positive colonies showed the correct band size. Positive clones were picked, plasmids were extracted, and second-generation sequencing was performed. Correctly sequenced plasmids and strains harboring the correct plasmid were obtained for phage construction.

Figure 24: The results of colony PCR. Positive colonies were expected at around 2kb. All colonies in the figure matched the expectation.

Figure 25: The Result of Plasmid Sequencing

1.2 Verification of Successful Phage Gene Replacement

We co-cultured the Escherichia coli MG1655 harboring pUC19-VO98_215 and the Escherichia coli MG1655 harboring pUC19-VO98_215(C-) with the WT T7 (wild-type T7 phage) until the majority of bacteria lysed.

Figure 26: The diagrams of Genome - Plasmid pUC19-VO98_215 and pUC19-VO98_215(C-) Homologous Recombination

After centrifugation, the supernatant was collected and filtered using a 0.22 μm membrane to obtain two T7 Mixes: T7 ∆gp17::VO98_215 and T7 ∆C-gp17::C-VO98_215. The phage genomes were extracted from the T7 Mixes using the phage genome extraction kit (TIANGEN, DP301).

Figure 27: WT T7 and Isolated phage genome

Using the isolated phage genome as a template, we performed PCR with four pairs of specific primers to detect distinct bands.

Figure 28: Binding Status of Specific Primers.
Primer VO98_215-up_test showed a band only in T7 ∆gp17::VO98_215 and other primers (VO98_215-down_test, VO98_215/(C-)-up, VO98_215/(C-)-down) produced bands in all strains except wild-type T7.

Following PCR amplification, we detected the target specific band via 0.8% agarose gel electrophoresis. Sequencing results confirmed both recombinant sequences were entirely correct. Additionally, PCR reactions using the WT T7 genome as a template yielded no detectable bands, ruling out background interference.Genomic PCR was performed in triplicate. When the annealing temperature was 58°C, there was no non-specific amplification and the replicate results were consistent.

Figure 29: The results of phage genomic PCR.
For primers VO98_215-up_test and VO98_215-down_test, the expected positive result band size is approximately 200bp.
For primer VO98_215/(C-)-up, the expected positive result band size is approximately 700bp.
For primer VO98_215/(C-)-up, the expected positive result band size is approximately 1400bp.

Figure 30: Partial sequencing of recombinant genome of T7 ∆gp17::VO98_215 and T7 ∆C-gp17:: C-VO98_215 .

2. Verification of Successful Heterologous Expression of the Tail Protein Gene

We successfully constructed the two His-tagged plasmids: pUC19-VO98_215-Chis and pUC19-VO98_215(C-)-Chis. Whole-plasmid sequencing results were accurate. WT T7 infected 200 mL each of log-phase MG1655/pUC19-T7arm-VO98_215-Chis and MG1655/pUC19-T7arm-VO98_215(C-)-Chis cultures, which were incubated at 37°C until clarification. Filter the supernatant through a 0.22 μm membrane, add the phage precipitation reagent (20% PEG8000, 2.5 M NaCl), and precipitate overnight at 4°C. Centrifuge, dry the pellet, resuspend in 1 mL of the loading buffer, and heat at 95°C for 10 min to obtain crude protein.

Add Ni-NTA Beads to a centrifuge tube. Centrifuge, discard the supernatant, and add the Lysing Buffer. Add the sample, seal the tube, and incubate at 37°C for 1 hour. After incubation, centrifuge, aspirate and discard the supernatant. Retain the supernatant as the flow-through for electrophoresis identification. Wash with the Wash Buffer, then elute with the Elution Buffer. Incubate at room temperature for 15 minutes, then centrifuge to collect the eluate.
Use bovine serum albumin (BSA) as the standard sample and determine the protein concentration of the initial eluted sample by the Coomassie Brilliant Blue staining.
The A595 value of the protein VO98_215-Chis is 0.246, and its concentration is calculated to be approximately 25.0μg/mL . The A595 value of the protein VO98_215(C-)-Chis is 0.358, and its concentration is calculated to be approximately 41.2μg/mL.

Figure 31: Standard Curve of Protein (left). The concentration of protein VO98_215-Chis and VO98_215(C-)-Chis (Right).

The remaining sample was concentrated using the ultrafiltration tube, achieving a 1000-fold concentration to obtain 30 μL of protein at a final concentration of approximately 20 μg/μL. After adding the loading buffer and mixing thoroughly, the sample was boiled at 95°C for 10 minutes to yield the purified protein sample.
The purified protein sample was used for Western blot (WB) analysis. A mouse anti-His-tag antibody (Abcam, ab18184; 1:2000 dilution in skimmed milk) served as the primary antibody, and a goat anti-mouse IgG antibody (1:2000 dilution in skimmed milk) was used as the secondary antibody. SDS-PAGE electrophoresis was performed using ACE 10% 1 mm precast gel and corresponding buffers. Adjust the loading volume to ensure 10 μg purified protein is loaded per lane. Run at 45 V until the bromophenol blue front reaches the end of the gel. Cut the gel from the rightmost three lanes for Coomassie Brilliant Blue R250 staining. Incubate at room temperature on a shaking incubator for 1 hour, then decolorize by boiling in a boiling water bath. Perform semi-dry transfer of the left gel for 12 minutes. Block with skim milk powder at room temperature on a shaking incubator for 1.5 hours. Incubate with the primary antibody (1:2000 dilution) overnight at 4°C. Wash the membrane five times with TBST for 10 minutes each. Incubate with the secondary antibody (1:2000 dilution) for 1 hour. Wash the membrane five times with TBST for 10 minutes each. Develop using the ECL chemiluminescent system with equal parts ECL Mix A and B.

Figure 32: Western Blot of phage WT T7, T7 ∆gp17::VO98_215 and T7 ∆C-gp17::C-VO98_215. The expected band size of the target protein is approximately 64 kDa, and it is present only in the phage T7 ∆gp17::VO98_215 and T7 ∆C-gp17::C-VO98_215.

Function of Recombinant Phages with Substituted Tail Protein

Verification of phage function encompasses both adsorption and infection aspects. The double-layer plates demonstrated that WT T7 and the recombinant phages (T7 ∆gp17::VO98_215 and T7 ∆C-gp17::C-VO98_215) failed to produce plaques directly on the DC3000 bacterial biofilms. The phenotype could not be directly observed. Therefore, we independently validated the functionality of the recombinant phage through both adsorption and infection pathways.

Figure33: Double-Layer Plating

1. Verification of Recombinant Phage Effective Adsorption on Pseudomonas syringae pv. tomato DC3000

Wild-type T7 phage infection of MG1655/pUC19-T7arm-VO98_215 and MG1655/pUC19-T7arm-VO98_215(C-) yielded the recombinant phages T7 ∆gp17::VO98_215 and T7 ∆C-gp17::C-VO98_215. After clarification, the lysate was treated with phage precipitation agents (20% PEG8000, 2.5 M NaCl), precipitated overnight at 4°C, and centrifuged. The pellet was resuspended in 1 mL SM buffer.

The resuspended sample yielded approximately 10¹¹ titer units for the titer determination.

Take 1 mL DC3000 (OD = 0.7), centrifuge at 7000xg for 5 min at 4°C, resuspend in 1 mL Ringer’ s solution. Mix 150 μL phage sample with 150 μL DC3000 and incubate for 10 min. Carbon films were floated from mica plates directly into the bacteria/phage mixtures and allowed to settle for 1 min, then stained with 1% uranyl acetate solution (prepared in deionized water) for 30 seconds. Images were acquired using a Hitachi TEM system set at an acceleration voltage of 80 kV.

Figure 34: Negative staining Transmission Electron Microscope detects the ability of phage with recombinant tail fiber to infect Pseudomonas syringae pv. tomato DC3000.
A: Dividing Pseudomonas syringae pv. tomato DC3000; B:Pseudomonas syringae pv. tomato DC3000; C: T7 bacteriophage; D: WT T7 can approach but cannot infect Pseudomonas syringae pv. tomato DC3000; E: T7 ∆gp17::VO98_215 infecting Pseudomonas syringae pv. tomato DC3000; F: T7 ∆C-gp17:: C-VO98_215 infecting Pseudomonas syringae pv. tomato DC3000.

The wild-type T7 phage (WT T7) can approach DC3000 but cannot adsorb to its surface. Furthermore, possibly due to the weak electrostatic interaction between WT T7 phage and DC3000, the number of WT T7 phages approaching a single bacterium is much lower than that of the other two recombinant phages under the same conditions.

Figure 35: WT T7 can approach but cannot infect DC3000

The recombinant phage T7 ∆gp17:::VO98_215 with full-length tail fiber replacement can approach and adsorb to the surface of DC3000. However, when compared with the infection result of phage T7 ∆C-gp17::C-VO98_215 on DC3000, it is found that the probabilities of T7 ∆gp17:::VO98_215 approaching and adsorbing to DC3000 are much lower than those of T7 ∆C-gp17::C-VO98_215.

Figure 36: T7 ∆gp17:::VO98_215 infecting DC3000 and the enlarged partial view.

The recombinant phage T7 ∆C-gp17::C-VO98_215 with C-terminal replacement of tail fiber protein has the highest efficiency of approaching and adsorbing to the surface of DC3000. Meanwhile, we observed that almost all DC3000 adsorbed by T7 ∆C-gp17::C-VO98_215 showed cell wall damage, which implies that the possibility of T7 ∆C-gp17::C-VO98_215 infecting DC3000 is very high.

Figure 37: T7 ∆C-gp17::C-VO98_215 infecting DC3000 and the enlarged partial view. The broken cell wall is circled with the dashed lines.

When the adsorption ratios of the three phages to DC3000 were calculated under the same scale bar, significant differences were found among their adsorption ratios. This indicates that the modification of tail fibers significantly improves the adsorption capacity of phages to DC3000, and the obtained recombinant phages have strong targeting ability.

Figure 38: phage adsorption ratio(Left); phage number heatmap(Right).

2. Verification of Tail Fiber-Replaced PLPs Effective Infection of DC3000

Green fluorescence was used as the forward screening marker. The ampicillin resistance expression frame in the packaging plasmid pUC19-packaging-PTetA-GFP was replaced with a kanamycin resistance expression frame to generate pUC19-packaging-PTetA-GFP-KanR. Subsequently, the packaging plasmid pUC19-packaging-PTetA-GFP-KanR carrying the fluorescent protein bjGFP was transformed into MG1655/pUC19-VO98_215 and MG1655/pUC19-VO98_215(C-), to construct dual-plasmid host strains E.coli MG1655/pUC19-VO98_215/pUC19-packaging-PTetA-GFP-KanR and E.coli MG1655/pUC19-VO98_215(C-)/pUC19-packaging-PTetA-GFP-KanR.
Infection of the dual-plasmid host with wild-type T7 phage yielded phage-like particles(PLPs) with capsids containing only pUC19-packaging-PTetA-GFP and tail proteins replaced. These PLPs were inoculated into DC3000 liquid culture, mixed, and spread onto the resistance plates for incubation at 28°C for 16 hours. Green fluorescent colonies were observed in DC3000, indirectly confirming the infectivity of the recombinant phage against DC3000.

Figure 39: LB plates spread with DC3000 after co-cultivation with PLPs

The experimental results demonstrate that the PLPs successfully targeted and infected pathogens beyond the native host spectrum through tail protein replacement and packaging with the packaging plasmids.