The Discovery And Identification Of A New Coliphage (EECP) Special For Genetically Engineered E. Coli

Bacteriophages, the simplest biological system of living nature, are the viruses of bacteria, fungi, actinomyces or spirochaetes. As an excellent model system and effective biological tool for the researches on interaction among DNA, RNA and protein, bacteriophages have been extensively applicated in many molecular biological researches, such as the reveal of basic vital phenomena, the investigation of biodiversity, the prevention and cure of refractory infection of drug-resistant bacteria, the treatment of environmental pollution, and so on. Consequently, microbiological and molecular biological researchers have focused on bacteriophages since their first discovery in 1915. The giant bacteriophage community has abundant biological diversity, and up to date, there are 484 completed phage genomes available in public databases.Genetically engineering bacteria, whose genome is artificially reconstructed with genetic engineering methods, also to be one of important tools and materials for molecular biological researches and gene engineering industrial productions. Among those successfully constructed genetically engineering bacteria, E.coli is used more frequently than others. As general bacteria, bacteriophage infection is the strongest threaten for the culture of genetically engineering bacteria. If those bacteria are attacked by relative phage, it would be a calamity because of the delay or completely stop of fermentation processes and research works. Up to now, 106 coliphages had been isolated and completely sequenced, but none is reported special for genetically engineered E.coli. EECP is new coliphage isolated occasionally in our lab. Our study was focused on the biological characterization and physicochemical stability of EECP. Results not only can characterize this temperate bacteriophage, but also can provide some useful information for setting up reasonable and effective strategies to handle its pollution in industries and at laboratories. The contents and results are showed as follows:1. EECP is new coliphage. Phage EECP was acquired occasionally from a conventional electroporatic transduction experiment which used a genetically engineered E.coli strains BL21 (DE3) as competent cells to express a protein. In order to discern whether EECP is a new bacteriophage, two enzymic fragments about 1700 bp and 700 bp of EECP genome cut with BamH I were separated, cloned and sequenced. Based on the analysis of sequencing results with Blastn software, no obvious homology was found between the sequences of known phages and EECP, so it is possible that EECP is new coliphage. Then the genome of EECP was sent out for complete sequencing. We also overthrew the guess that EECP was lambda phage based on the compare of genomic cleavage map of EECP and lambda phage. According to all results above, we concluded that EECP is a new coliphage and it is worthy of deeply research and complete genomic sequencing.2. EECP is a genetically engineered E.coli phage. EECP could infect 10 of 11 tested genetically engineered E.coli strains, including BL21 (DE3), DH10B, DH5α, JM109, M15, Rosetta-gami, Rosetta, S17-1, S17-1λPir+ and TOP10. Among the 10 sensitive genetically engineered E.coli strains, 8 were derivatized from E.coli K-12 and 2 from E.coli B strain.3. EECP is a bacteriophage with possible moving capbility. Electron microscopy revealed that EECP has an isometric head (about 79-83 nm in diameter) and a long exceptional anfractous tail (about 180-220 nm in length and 7-8 nm in width). The tail of EECP seems to be flexible and can swing as bacterial flagella, which make it possible that EECP can move depend on the flap of the tail. Furthermore, we also found curiously that some plaques had a long tailing beside them which seemed to be the trace of the directional moving of EECP. According to those findings, we thought EECP as a bacteriophage that can move. But because of the deficiency of proper technique, we could not verificate the moving of EECP with experiments.4. The biological characterization of EECP.①EECP is a temperate coliphage. After be incubated together with BL21 (DE3) at double-layered LB agar plate overnight, EECP would form plaques (ca 1-2 mm in diameter) with a limpid center surrounded by a translucent loop. The edge of the loop was ambiguous and less keen.②Judged from its morphous, nucleic acid and the less of lipidic membrane, EECP belongs to the myoviridae family.③The optimal multiplicity of infection (MOI) of EECP on BL21 (DE3) is about 0.01.④One-step growth kinetics of the phage showed that the latent period is about 10-15 min, the rise period is about 30-40 min, and the average burst size is about 375±43 phage particles per infected cell.⑤Based on restriction enzyme analysis, the genome of EECP was found to be a circular double-stranded DNA molecule of about 45 kb.⑥The capsid of EECP phage was composed of at least 9 structural proteins, with the molecular weight of 136.38,91.47,63.91,45.20,36.66,29.19,24.20,21.88 and 15.84 kD (called protein #1 to #9 in turn), among which the protein of 45.20 kD was predominant. N-terminal amino acids of some structural protein (#2,#3,#4,#6,#9) are"ADQAA","TVNVD","SLTVF","GYQLP"and"MLDSI"individually.⑦Based on the bioinformatics analysis of the 40061 bp of EECP genome, 225 ORFs longer than 100bp were found out by ORF finder software at NCBI website and 59 predicted genes were recognized using GeneMark and Softberry programs. The G+C content of EECP genome is 54.65%.⑧5 EECP capsid proteins of 136.38 kD, 91.47 kD, 63.91 kD, 29.19 kD, 15.84 kD were considered to be the coding products of gene orf24, orf30, orf52, orf33, orf46.5. The physical and chemical stability of EECP.①EECP could survive from high thermal treatments. Heating at 90°C for 45 min was insufficient for complete inactivation of high-titer phage suspensions, but no active phage was checked after treated at 95°C for 5 min.②Ethanol also not to be a potent disinfectant for EECP, even the ethanol of its effective concentration (75% and 100%, vol/vol) was failing to deactivate all phage.③EECP Phage could maintain its infectivity at alkaline condition at range of PH 5 to11 for 60 min, but strong acid (PH 2-3) killed phage once it was added in. Furthermore, EECP was more sensitive to tort state at 37°C than at 25°C.④the presence of Na+ could weaken the lytic capability of EECP, and the lytic ability might be further attenuated when Ca2+ and Mg2+ were also added in..