Figure 1.
Genomic structures of our synthetic bacteriophages.
(A) The S13 genome is 5386 nucleotides long and it codes for 11 genes. The four mutation sites are located at the F coding region. (B) S13/G4 chimeric recombinant genome. This new phage genotype consists of the S13 genome with its allele of F replaced by the homologue from G4. Blue areas represent regions of S13 phage and the green area represents the F gene of G4 phage. The label “G:F” represents the untranslated region between genes G and F and the other label “F:J” represents the untranslated region between genes F and J.
Figure 2.
Images of the plaques (8 h, 37°C, 10 cm Petri dish).
A. wt-S13 B. m1-S13 C. m2-S13 D. S13/G4 chimera.
Figure 3.
Test for the presence of engineered HaeIII genetic markers in m1-S13.
Lane 1 and lane 8 contained the D2000marker. As expected, only a single band of 628 bp was observed when PCR products of wt-S13 were digested with HaeIII, due the absence of the genetic marker (lane 2 and 3). A single band of 628 bp was observed when PCR products of m1-S13 were not digested with HaeIII (lane 4 and 5). Electrophoretic analysis of the PCR products of the m1-S13 showed the presence of 3 bands (316nt,192 nt and 120nt) after digestion with HaeIII, an observation indicating that the synthetic phage contained the engineered markers(lane 6 and 7). The values to the left are molecular sizes in base pairs (bp).
Figure 4.
A phylogenetic tree constructed by using the maximum parsimony method.
Sequence cluster analysis based on the whole-genome sequences of our synthetic phages and other related phage sequences obtained from the GenBank database.
Figure 5.
Biological characteristics of the synthetic phages.
(A)Time courses of host cell lysis by synthetic phages. A growth curve for E. coli C with no treatment was reproduced for comparison. (B)Temperature stability test. Phages were incubated under different temperature values for 30 min before determining the percentage of surviving infectious phages. (C) pH stability test. Phages were incubated under different pH values for 30 min before determining the percentage of surviving infectious phages. (D) Inactivation of phages by ultraviolet irradiation. Samples were taken at different time intervals to calculate the percentage of surviving infectious phages. Results were shown in Mean ± S.D. from three independent experiments.
Table 1.
Adsorption rates of the synthetic phages.