Team:Mingdao/Proof Of Concept

iGEM 2021: Mingdao

Proof Of Concept



  After visited and discussed with a Taiwan’s phage expert, Prof. Chih-Hsin Hung, at the Department of Chemical Engineering of I-Shou University in Kaohsiung in the southwest of Taiwan, we got the phage hunting protocol and were confident to search Salmonella phages. We successfully isolated Salmonella phages (Fig. 1) from Liuchuan River near our campus, Mingdao High School, in Taichung City using the procedure in the following and recording in the video.

Figure 1 |The phage plaques on a Salmonella culture


    ↓ collect the sample from a river
    ↓ pass through a filter paper
    ↓ add Salmonella and LB powder
    ↓ culture for 4 hours
    ↓ centrifuge and filter with a membrane of 0.45 μm pore size
    ↓ make 0.75% soft agar containing the filtrates and Salmonella
    ↓ overlay on a LB agar plate
    ↓ overnight culture
    ↓ observe the plaques


  Two of the plaques were further determined for the specificity to Salmonella. The isolated phages (named ST1 and ST2) and E. coli phages (T4 and T7) were tested on Salmonella Typhimurium LT2 and E. coli DH5 α, respectively. The data in Fig. 2 represented the phages #ST1 and #ST2 can specifically infect and lyse Salmonella as plaques instead of E. coli. And E. coli T4 and T7 phages can't infect Salmonella as experimental controls.

Figure 2 |Plaque assays on Salmonella Typhimurium LT2 or E. coli with the isolated phages #ST2 and #ST2 or E. coli phages T4 and T7, respectively.


  The genome of Salmonella phage #ST1 was extracted and engineered to carry phi29 DNA polymerase gene through the Tol2 transposon system.

  T7-His-Tol2 transposase was expressed in TXTL using IPTG-induced E. coli Rosetta 2(DE3) extracts, followed by purification with Nickel column. The DNA fragment of Tol2 transposable element carrying ldhp-Phi29 DNA polymerase-Tr (Ф29/Tol2) was amplified by PCR. The extracted Salmonella phage genome and the Ф29/Tol2 DNA fragment were incubated with Tol2 transposase at 30°C for 2 hours. The recombinant phage genome was subjected to TXTL based on the work of Jonghyeon Shin1, where T7 phage genome can be replicated, synthesized, and assembled in a single cell-free reaction. The infectious phage of Salmonella phage #ST1::Ф29 made by TXTL using Salmonella extracts was tested on plaque assay with a culture of Salmonella on the LB agar plate. As shown in Fig. 3b, visible different sizes of plaques were formed on the agar plate, demonstrating infectious phages were produced in our TXTL system. As a control, the phage gDNA without TXTL reaction displayed no plaques (Fig. 3a), indicating the live phages from TXTL are not from the contaminated DNA in the process of genomic DNA extraction.

Figure 3 |The recombinant phage synthesis in TXTL. Plaques were formed on a lawn of Salmonella culture on the LB agar plate from TXTL reaction (b) compared to no plaques without TXTL reaction (a).

  Dozens of plaques were screened by PCR with phi29 DNA polymerase gene-specific primers. A representative result on DNA gel electrophoresis was shown in Fig. 4, in which the successful Ф29/Tol2 insertion (Salmonella phage::Ф29 DNA polymerase, or #ST1::Ф29 for short) can be amplified by PCR with either Tol2 transposable element-specific primers or phi29 DNA polymerase-specific primers, compared to no PCR products from wild-type Salmonella phage #ST1, showing the success of our Salmonella phage engineering with phi29 DNA polymerase gene.

Figure 4 |Salmonella genome were checked by PCR with Tol2 transposable element-specific primers (lanes 1, 2) or with phi29 DNA polymerase gene-specific primer set 1 (lanes 3, 4) or set 2 (lane 5, 6). The odd numbers refer to Salmonella phage::Ф29 DNA polymerase (#ST1::Ф29), and the even numbers refer to wild-type Salmonella phage #ST1. The gel electrophoresis was performed on a 1% agarose gel with a 1kb DNA ladder.


  We amplified the Salmonella phage #ST1::Ф29 in the culture of Salmonella Typhimurium LT2, then harvested, centrifuged, purified, and titrated the phage concentration by a serial dilution test in plaque assay. The average titers of phage #ST1::Ф29 achieved around 109 PFU/ml (Fig. 5).

Figure 5 |Plaque assay with a serial dilution of the amplified engineered Salmonella phage #ST1::Ф29 DNA polymerase. The data were from 3 independent phage amplification procedures. The plaque images were the representative results.


  An overnight culture of Salmonella Typhimurium LT2 (~109 cells/ml) were infected by the engineered reporter phage #ST1::Ф29 at MOI=0.1 to produce phi29 DNA polymerase. The lysates were collected after 2 hr or 4 hr of treatment, and then subjected to RCA test. The lysate of phage-infected Salmonella at 2 hr can induce strong RCA reaction (24-fold change) comparable to 2.5U of a commercial phi29 DNA polymerase (NEB) in Fig. 6. Surprisingly, the lysate at 4 hr can not trigger any signal in RCA, suggesting a quick decay of phi29 DNA polymerase in the phage-infected bacterial lysates.

Figure 6 |RCA assay with NEB phi29 DNA polymerase or the Salmonella (109 cells/ml) lysates infected by #ST1::Ф29 at MOI=1 which were collected at 2 hr or 4 hr post infection. The fold changes were calculated by the fluorescence intensity of EvaGreen DNA binding of RCA materials without phi29 DNA polymerase as a background control. RCA was performed at 30°C for 1 hr.


  We are wondering whether the time of bacterial lysis by phage infection affects the stability of phi29 DNA polymerase protein. An isolated phage may be featured by a latent time (phage generation time in a bacterial cell) and a burst size (numbers of phage produced per bacterial cell). And the latent time and burst size are in a relationship in terms of bacterial density2 and MOI of phage infection3.

  Therefore, we performed the experiment to figure out the latent time and burst size of our Salmonella phage #ST1::Ф29 and the time course of RCA signals during phage infection in Salmonella. 105 cells/ml of Salmonella were infected by phage #ST1::Ф29 at MOI=1. The lysates collected by an interval of 5 min until 60 min and subjected to plaque assays and RCA test. As Fig. 7 demonstrated, the phages were released at around 40 min (latent time) to a plateau level with a burst size of average 98.4 ± 14. Interestingly, the RCA signal increased dramatically at 35 min, achieved a high level around 40-45 min, and dropped significantly thereafter, that are consistent with our speculation of the correlation between phage lysis time (latent time) and phi29 DNA polymerase protein functionality.

Figure 7 |Salmonella phage #ST1::Ф29 latent time (min) and burst size (PFU per infected cell, the left Y axis) at MOI=1, and the relationship to RCA assay (fold change, the right Y axis). Phage-infected Salmonella lysates were harvested for 1 hour at an interval of 5 min. The lysates were subjected to plaque assays and RCA reaction followed by stained with EvaGreen dye. The burst sizes were counted by numbers of plaques. The RCA signal were read at Ex/Em=500/530 nm and divided by the background level without phi29 DNA polymerase.


  Imagine an application of real Salmonella diagnosis in a food or drink. A contaminated sample collected in a large volume may find 1-100 CFU/ml of bacteria4. Large volume and bacterial density are key parameters to affect the result of Salmonella detection with our product.

  We used the mathematical modeling to simulate the latent time of our isolated engineered phage in terms of bacterial density based on the study of Stephen T. Abedon, et al5. Go to our page of (MODEL for a detail. For the low density of bacteria (1-100 CFU/ml) usually observed in the contaminated food, our mathematical model predicts the optimal latent time of phage infection is between 16.545 and 24.427 min. Therefore, we thought 25 min are the best time for RCA assay.

  To overcome the large volume of a sample, we were inspired by Nickel column purification, in which His-tagged phi29 DNA polymerase were bound. Therefore, we think this method may enrich the His-phi29 DNA polymerase from the sample of large volume. Go to our page of (HARDWARE for such a design. And the schematic diagram was shown here.

  To examine the feasibility, we made a serial dilution of Salmonella from 107 to 103 cells in a beaker of 500ml water and prepared the water without bacteria as a control. The water were mixed with Salmonella phage #ST1::Ф29 at the concentration of 105 PFU/500ml at room temperature for 25 min. The 3D-printed Luer locker embedded a mini Ni-column was assembled onto a syringe, followed by repeatedly drawing up and pushing back the water in order to pass through the Ni-column. Then, the RCA materials were drawn onto the Ni-column. If His-phi29 DNA polymerase is present, the RCA reaction may be turned on. After 30 min incubation for RCA reaction, the mixtures were push back into a well of a 96-well black plate containing EvaGreen Dye in a total volume of 50 μl. The fluorescence signals were measured at Ex/Em=500/530 nm. Significant RCA signals began to appear in 2x102 bacterial cells/ml (Fig. 8). 20 cells/ml can be detected with a slight enhanced signal that is able to be distinguished from the background. However, we can’t measure the cell density under 10 cells/ml of a liquid to be examined.

Figure 8 |Salmonella test at various concentrations between 2-2x104 cells/ml in 500ml water with engineered Salmonella phage carrying His-phi29 DNA polymerase gene at the concentration of 200 PFU/ml. RCA was performed on the embedded Ni-column in a 3D-printerd Luer lock adapter. The amplified DNAs were stained with EvaGreen Dye and measured at Ex/Em=500/530 nm in a microplate reader (BioTek Synergy H1).


  We demonstrated a proof of concept that it is possible to detect Salmonella by an isolated genetically engineered Salmonella-specific phage carrying phi29 DNA polymerase gene. The His-tagged phi29 DNA polymerase can be enriched from a large volume of a sample onto the homemade mini Ni-column in a Luer-lock adaptor format, where isothermal RCA may be triggered in the presence of the extraordinarily processive phi29 DNA polymerase at room temperature in a time as short as within 30 min.

  Bacteriophages possess features that can produce large amounts of phage progeny (the burst size, usually dozens to hundreds PFU per infected bacterium) to release by killing bacterial host in a short time (the latent time, usually 20 min to 1 hr). We mathematically modeled the latent time (min) as a function of cell density (cells/ml) and applied it to simulate a possible real condition using reporter phage to detect Salmonella in a poisoning case. We predicted the optimal bacterial lysis time (latent time) for RCA is between 16.545 and 24.427 min for 1 to 100 cells/ml of bacteria.

  Finally, we developed a hardware of 3D-printed Luer-lock adapter containing mini Ni-column. We used it to measure the Salmonella in 500 ml of water. We can detect 200 bacterial cells/ml within 1 hour. About 20 cells/ml of bacteria may be the most probable limit of detection in our device that generated a slight but distinguishable signal compared to the background level of no bacteria control.

  Compared to traditional Salmonella tests and published engineered reporter phage, we think our product is better than traditional method in term of test time, and our product is comparable and competitive in terms of test limit and time to the current designer phages carrying reporter genes6,7,8 (Table 1).


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