Gene synthesis is an enabling technology that supports the development of synthetic biology. The existing approaches for de novo gene synthesis generally have tedious operation, low efficiency, high error rates, and limited product lengths, being difficult to support the huge demand of synthetic biology. The assembly and error correction are the keys in gene synthesis. This study first designed the oligonucleotide sequences by reasonably splitting the virus genome of approximately 10 kb by balancing the parameters of sequence design software ability, PCR amplification ability, and assembly enzyme assembly ability. Then, two-step PCR was performed with high-fidelity polymerase to complete the de novo synthesis of 3.0 kb DNA fragments, and error correction reactions were performed with T7 endonuclease Ⅰ for the products from different stages of PCR. Finally, the virus genome was assembled by 3.0 kb DNA fragments from de novo synthesis and error correction and then sequenced. The experimental results showed that the proposed method successfully produced the DNA fragment of about 10 kb and reduced the probability of large fragment mutations during the assembly process, with the lowest error rate reaching 0.36 errors/kb. In summary, this study developed an efficient de novo method for synthesizing a viral genome of about 10 kb with T7 endonuclease Ⅰ-mediated error correction. This method enabled the synthesis of a 10 kb viral genome in one day and the correct plasmid of the viral genome in five days. This study optimized the de novo gene synthesis process, reduced the error rate, simplified the synthesis and assembly steps, and reduced the cost of viral genome assembly.
Genome, Viral/genetics* ; Polymerase Chain Reaction/methods* ; DNA, Viral/genetics* ; Bacteriophage T7/enzymology* ; Synthetic Biology/methods*

OBJECTIVES: The emergence of resistant bacteria is being increasingly reported around the world, potentially threatening millions of lives. Amongst resistant bacteria, methicillin-resistant Staphylococcus aureus (MRSA) is the most challenging to treat. This is due to emergent MRSA strains and less effective traditional antibiotic therapies to Staphylococcal infections. The use of bacteriophages (phages) against MRSA is a new, potential alternate therapy. In this study, morphology, genetic and protein structure of lytic phages against MRSA have been analysed. METHODS: Isolation of livestock and sewage bacteriophages were performed using 0.4 μm membrane filters. Plaque assays were used to determine phage quantification by double layer agar method. Pure plaques were then amplified for further characterization. Sulfate-polyacrylamide gel electrophoresis and random amplification of polymorphic DNA were run for protein evaluation, and genotyping respectively. Transmission electron microscope was also used to detect the structure and taxonomic classification of phage visually. RESULTS: Head and tail morphology of bacteriophages against MRSA were identified by transmission electron microscopy and assigned to the Siphoviridae family and the Caudovirales order. CONCLUSION: Bacteriophages are the most abundant microorganism on Earth and coexist with the bacterial population. They can destroy bacterial cells successfully and effectively. They cannot enter mammalian cells which saves the eukaryotic cells from lytic phage activity. In conclusion, phage therapy may have many potential applications in microbiology and human medicine with no side effect on eukaryotic cells.
Agar ; Bacteria ; Bacteriophages ; Caudovirales ; Classification ; DNA ; Electrophoresis ; Eukaryotic Cells ; Head ; Humans ; Livestock ; Membranes ; Methicillin Resistance ; Methicillin-Resistant Staphylococcus aureus ; Methods ; Microscopy, Electron, Scanning Transmission ; Microscopy, Electron, Transmission ; Sewage ; Siphoviridae ; Staphylococcal Infections ; Tail

We have examined isolation and identification protocols for three virus simulant candidates to biological warfare agents. MS2 phage, a simulant for yellow fever virus and Hantaan virus, was propagated using as a host an E. coli strain with F pilus. MS2 phage genome was examined by reverse transcription and polymerase chain reaction (RT-PCR). Coat protein of the phage preparation was examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometric analysis. Cydia pomonella granulosis virus (CpGV) is a virus simulant candidate to smallpox virus. CpGV was isolated from a commercialized CpGV pellet. In this study, we developed new isolation and identification protocols for CpGV. One disadvantage of using CpGV is that it is not easy to determine viability of the virus. Here, we have included T4 phage as an alternative. We established a high titer production protocol and developed an easy genome identification protocol that does not require purified phage DNA. Stability of these virus preparations was also examined under various storage conditions. When the virus preparations were not subjected to freeze drying, MS2 phage was most stable when it was stored in liquid nitrogen but unstable at 4℃. In contrast, T4 phage was most stable when it was stored at 4℃. CpGV was stable at −20℃ but not at 4℃. Stability during or after freeze drying was also investigated. The result showed that 70~80% MS2 survived the freeze drying process. In contrast, only about 15% of T4 phage survived during the freeze drying. CpGV was found to be degraded during freeze drying.
Bacteriophage T4 ; Bacteriophages ; Biological Warfare Agents ; DNA ; Electrophoresis ; Freeze Drying ; Genome ; Granulovirus ; Hantaan virus ; Levivirus ; Nitrogen ; Polymerase Chain Reaction ; Reverse Transcription ; Variola virus ; Yellow fever virus

This study aimed to develop a new multiplex real-time PCR detection method for 3 species of waterborne protozoan parasites (Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis) identified as major causes of traveler's diarrhea. Three target genes were specifically and simultaneously detected by the TaqMan probe method for multiple parasitic infection cases, including Cryptosporidium oocyst wall protein for C. parvum, glutamate dehydrogenase for G. lamblia, and internal transcribed spacer 1 for C. cayetanensis. Gene product 21 for bacteriophage T4 was used as an internal control DNA target for monitoring human stool DNA amplification. TaqMan probes were prepared using 4 fluorescent dyes, FAM™, HEX™, Cy5™, and CAL Fluor Red® 610 on C. parvum, G. lamblia, C. cayetanensis, and bacteriophage T4, respectively. We developed a novel primer-probe set for each parasite, a primer-probe cocktail (a mixture of primers and probes for the parasites and the internal control) for multiplex real-time PCR analysis, and a protocol for this detection method. Multiplex real-time PCR with the primer-probe cocktail successfully and specifically detected the target genes of C. parvum, G. lamblia, and C. cayetanensis in the mixed spiked human stool sample. The limit of detection for our assay was 2×10 copies for C. parvum and for C. cayetanensis, while it was 2×10³ copies for G. lamblia. We propose that the multiplex real-time PCR detection method developed here is a useful method for simultaneously diagnosing the most common causative protozoa in traveler's diarrhea.
Bacteriophage T4 ; Cryptosporidium parvum ; Cryptosporidium ; Cyclospora ; Diagnosis ; Diarrhea ; DNA ; Fluorescent Dyes ; Giardia lamblia ; Giardia ; Glutamate Dehydrogenase ; Humans ; Limit of Detection ; Methods ; Multiplex Polymerase Chain Reaction ; Oocysts ; Parasites ; Real-Time Polymerase Chain Reaction

Staphylococcal nuclease A (SNA) may be used to produce bacterial ghosts for further inactivation of host bacteria and elimination of residual genetic materials. It is still controversial if SNA without signal peptide can be secreted to extracellular matrix and if fusion with other peptide is required for its function in the cytoplasm of host bacteria. To clarify this dispute, a series of temperature-inducible plasmids carrying SNA alone or SNA fused with partial sequences of λ phage cro gene (cSNA) or Mycobacterium tuberculosis urease gene (uSNA) were constructed and evaluated in Escherichia coli. Results show that the percentages of inactivated E. coli by SNA, cSNA and uSNA after 4 h of induction were 99.9%, 99.8% and 74.2%, respectively. Moreover, SNA and cSNA in the cytoplasm of host bacteria were initially detectable after 30 min of induction, whereas uSNA was after 1 h. In comparison, SNA and cSNA in culture supernatant were initially detectable 1 h later, whereas uSNA was 2 h later. The nuclease activity in the cytoplasm or supernatant was ranked as follows: SNA > cSNA > uSNA, and the activity in the supernatant was significantly lower than that in the cytoplasm. Furthermore, host genomic DNA was degraded by SNA or cSNA after 2 h of induction but not by uSNA even throughout the whole experiment. In conclusion, this study indicates that SNA, cSNA and uSNA expressed in host bacteria all have nuclease activity, the enzymes can be released to culture media, and fusion with exogenous peptide negatively reduces the nuclease activity of SNA.
Bacteriolysis ; Bacteriophage lambda ; DNA ; chemistry ; Escherichia coli ; Genetic Vectors ; Micrococcal Nuclease ; chemistry ; Plasmids ; Protein Sorting Signals

To construct a recombinant T7 phage expressing matrix protein 2 ectodomain (M2e) peptides of avian influenza A virus and test immunological and protective efficacy in the immunized SPF chickens. M2e gene sequence was obtained from Genbank and two copies of M2e gene were artificially synthesised, the M2e gene was then cloned into the T7 select 415-1b phage in the multiple cloning sites to construct the recombinant phage T7-M2e. The positive recombinant phage was identified by PCR and sequencing, and the expression of surface fusion protein was confirmed by SDS-PAGE and Western-blot. SPF chickens were subcutaneously injected with 1 X 10(10) pfu phage T7-M2e, sera samples were collected pre- and post-vaccination, and were tested for anti-M2e antibody by ELISA. The binding capacity of serum to virus was also examined by indirect immunofluorescence assay in virus- infected CEF. The immunized chickens were challenged with 200 EID50 of H9 type avian influenza virus and viral isolation rate was calculated to evaluate the immune protective efficacy. A recombinant T7 phage was obtained displaying M2e peptides of avian influenza A virus, and the fusion protein had favorable immunoreactivity. All chickens developed a certain amount of anti-M2e antibody which could specially bind to the viral particles. In addition, the protection efficacy of phage T7-M2e vaccine against H9 type avian influenza viruses was 4/5 (80%). These results indicate that the recombinant T7 phage displaying M2e peptides of avian influenza A virus has a great potential to be developed into a novel vaccine for the prevention of avian influenza infection.
Animals ; Antibodies, Viral ; blood ; Bacteriophage T7 ; genetics ; immunology ; metabolism ; Chickens ; Enzyme-Linked Immunosorbent Assay ; Gene Expression Regulation, Viral ; Immunization ; Influenza A virus ; genetics ; immunology ; Influenza Vaccines ; immunology ; Influenza in Birds ; immunology ; metabolism ; prevention & control ; Peptides ; genetics ; immunology ; metabolism ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; Specific Pathogen-Free Organisms ; Viral Matrix Proteins ; genetics ; immunology ; metabolism

To confirm the hypothesis that the high frequency sequences of high throughput sequencing are the terminal sequences of the bacteriophage genome. An adaptor of specific sequence was linked to the end of the bacteriophage T3 genomic DNA, which was then subject to high throughput sequencing; as a control, the same T3 genomic DNA without adaptor was also analyzed by high throughput sequencing. The sequencing results were examined with bioinformatics software. Similar high throughput sequencing technique was applied to analyze the genomic sequence of N4-like bacteriophage IME11. Bioinformatics study showed that the sequences tagged with adaptors were consistent with the high frequency sequences without adaptor labeling. Our analysis also indicated that the end of the T4-like phage genome had specific sequences instead of random sequences, disagreeing with the previous assertion. Evidences were provided that N4-like bacteriophage had a particular terminal sequence: the left end of the genome was unique while the right end was permuted. The high throughput sequencing technique was convenient and practical to be used to simultaneously detect the terminal sequence and the complete sequence of bacteriophage genome.
Caudovirales ; genetics ; Computational Biology ; Genome, Viral ; High-Throughput Nucleotide Sequencing ; methods

Bacterial ghost is intact envelope of Gram-negative bacteria, which is produced by the function of the lysis gene E from bacteriophage PhiX174. The expression of the lysis gene E is usually controlled by the thermosensitive lambdapL/pR-cI857 promoter. In this study, we described a mutation (T --> C) at the ninth nucleotide of the OR2 in the lambdapR promoter of the lambdapL/pR-cI857 system by overlap PCR. The bacteriolytic assay showed that the mutation in the lambdapL/pR-cI857 system enhanced the temperature of repressing the expression of gene E up to 37 degrees C. The lysis efficiency of altered lambdapR promoter in Escherichia coli DH5a and avian pathogenic E. coli DE17 was up to 99.9%. The expanded range of temperature will benefit for the production of bacterial ghost.
Bacteriolysis ; physiology ; Bacteriophage lambda ; genetics ; Base Sequence ; Cell Membrane ; physiology ; DNA, Bacterial ; analysis ; Escherichia coli ; genetics ; growth & development ; physiology ; virology ; Gene Expression Regulation ; genetics ; Molecular Sequence Data ; Mutation ; Promoter Regions, Genetic ; genetics ; Viral Proteins ; genetics ; metabolism

Serratia marcescens jn01 was employed as the host for the isolation of phages from environmental sewage. One strain of phage named SmPjn was purified by picking transparent plaque with 2mm diameter and clear edge on the double-layer agar repeatedly. Electron micrographs indicated that the phage head was icosahedral with head size and tail length of (58 +/- 2.16) x (55 +/- 0.47) nm and (7 +/- 1.25) nm, respectively. On the basis of the morphology, this phage belongs to the family Podoviridae. Host-range determination revealed that the phage was capable of infecting the other two isolates of S. marcescens, P25 and CMCC41002. The optimal multiplicity of infection was 1. A one-step growth curve of SmPjn indicated that the latent period and burst size were estimated at 50 min and 1,125 pfu/cell, respectively . Genomic DNA of SmPjn was above 27kb in size and could be digested by Hind Ill and EcoR I into 11 and 9 visible fragments after electrophoresis, respectively. A novel Podoviridae-phage infecting S. marcescens was firstly reported in China.
China ; DNA, Viral ; genetics ; isolation & purification ; metabolism ; Host Specificity ; Podoviridae ; genetics ; growth & development ; isolation & purification ; Restriction Mapping ; Serratia marcescens ; physiology

We isolated a novel Enterobacteria phage IME08 from hospital sewage, then confirmed it was a double-stranded DNA phage by digesting its genetic material with DNase I, RNase A and several restriction endonucleases respectively. BLAST results of random fragments generated by a random PCR cloning method revealed that it belonged to T4-like virus. We subsequently determined the host recognizing genes (g37 and g38) sequence with a PCR-based "genome jumping" protocol based on highly conserved region at 5' terminus of g37 from four other T4-like Bacteriophages (T4, JS98, T2 and K3). These molecular biological methods enabled us to readily characterize the bacteriophage and efficiently determine the sequence of the genes of interest based on very limited conserved sequence information.
Bacteriophage T4 ; genetics ; isolation & purification ; Cloning, Molecular ; DNA, Viral ; genetics ; Escherichia coli ; genetics ; virology ; Genome, Viral ; genetics ; Host Specificity ; genetics ; Polymerase Chain Reaction ; methods