Biological Characters And The Technique Exploring Of Genome Remolding Of Pseudomonas Aeruginosa Phage PaP3

Bacteriophages are virus of bacteria.They are recognized to be the most numerous life-form in the biosphere.Since they were discoveried in 1915,bacteriophages have been at the forefront of molecular biological research,both as model systems and biological tools for the study and manipulation of bacterial genes.Many general concepts of contemporary biology were derived from work with phages.Lysogenic phages and the others mobile DNA elements(transposons,integrative plasmids,pathogenicity islands,and IS elements) are important vehicles for the gene lateral transfer between bacterial strains,then bring the biological diversity.They play an important role in bacteria evolution,antibiotic-resistant and pathogenicity,and also enhance the fitness of the bacteria.Deep insight into the genetic background of phages is very important for no matter investigating the interaction of phages and their host bacteria,and revealing the biological diversity mechanism resulting from gene horizontal transfer caused by phages.Currently,phage research is becoming a hot spot of microbiology.In addition,multidrug-resistant bacteria are becoming serious increasingly because of antibiotic abuse,and as the virus of infecting bacterias specifically, the bacteriophages in controlling bacterial infection are presented,some of which show therapeutic promise.Today,multivalency phages or engineering remolded phages have the optimistic perspective in antibacterial therapy.For this reason,it is very necessary to establish a method to remold phage genome.In this study,we focused on the biological characters of Pseudomonas aeruginosa phage PAP3,and remolded genome of Pseudomonas aeruginosa phage PaP3 through restriction endonucleasing,PCR deletion and religasing,at last,the remolded genome was electroporated into host cell.The contents and results are as follows:1.The biological characters of PaP3. â‘ Electron microscopy revealed that the Pseudomonas aeruginosa phage PaP3 has an isometric head(about 65nm in diameter) and a short tail,and it belongs to pedoviridae families.â‘¡Bacteriophage PaP3 is a temperate phage of Pseudomonas aeruginosa strain PA3, the plaques of PaP3 are semitransparent and their sizes are about 0.2cm in diameter.â‘¢The one-step growth curve of PaP3 revealed that latent periods is about 20min,the rise periods is 60min,and the average burst size is about 31 phage particles per infected cell. The data demonstrate one life circle of PaP3 from phage absorption to offspring particle releasing.â‘£0.001 MOI-infected host bacteria is gained the highest phage offsprings,about 4.0×10¹⁰pfu/ml.So the optimal multiplicity of infection of PaP3 is 0.001.⑤Rate constant of reaction between antiserum and PaP3 is 262,it means that the antigenicity of PaP3 is moderate.Cross neutralization test among three phages indicates there is little dependablity among these three phages.2.Genomic remolding ofPseudomonas aeruginosa phage PAP3.PaP3 genomic DNA was cut by Pacâ… ,Sphâ… and Sacâ…¡step-by-step,and all the fragments were reclaimed using Gel extraction kit.The fragment contain tRNA genes was used as template to perform PCR deletion reaction.PCR product was cloned into pBSK vector and identified by sequencing.Finally,remolded genome ofPseudomonas aeruginosa phage PaP3 was obtained by ligating propotional fragment.3.Tansforming remolded PaP3 genomic DNA into host cells by electroporation.â‘ Conditions of PaP3 genomic DNA electroporated into host cells.A P.aeruginosa strain PA3 was used as the recipient strain.Effects of growth stage of the strain, electroporation medium,Erythromycin concentration,osmotic pressure,field strength, DNA concentration and competent cells density on electroporation rate of PaP3 genomic DNA were observed under different conditions.It was showed that the highest transformation rate of electroporation was 2.1×10³pfu/μgDNA under the condition in which the cells were cultured to stationary phase in LB adding 50μg/ml Erythromycin and concentrated to about 10¹¹ cells/ml with 100mM sucrose at 25℃,the mixture of the competent cells and PaP3 DNA was eletroporated at 12kV/cm,300Ω,25μF.The results will be very helpful for the study of genomic function of Pseudomonas aeruginosa phage PAP3. â‘¡Electroporation of remolded PaP3 genome.The rernolded genome DNA was electroporated under the condition above metioned,but none of infective phage was obtained finally.In conclusion,the basic biological characters of Pseudomonas aeruginosa phage PaP3 was investigated,and the conditions of P.aeruginosa phage PaP3 genomic DNA electroporated into host cells was also explored.To study PaP3 tRNA genes function,four tRNA genes were deleted using PCR method.Though none of infective phage was obtained after remolded PaP3 genome had been electroporated,it will be a new approach to research genes function of phage.