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Innovation education was introduced in medical microbiology teaching practice,including updating the innovative education concept,reforming teaching methods and means,constructing the teaching content system and practice platform adapting to innovation education.
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Objective To study the technique of transposon mutagenesis involving the use of Mini-Tn5 on a suicide plasmid for Citrobacter freundii (C. freundii). Methods A conjugation method was used in transposon mutagenesis. The stability of exconjugants was confirmed by series passages under nonselective condition. Detection of anti-ampicillin resistance and purification of the plasmid were conducted for excluding the possibility of plasmid-encoded antibiotic resistance. All exconjugants were screened for auxotrophic mutants and swarming motility mutants. Results Analysis of the generated exconjugants indicated that Mini-Tn5 Km was transposed in C. freundii at a high frequency (ca. 9.2?10 -6). The mutants were stable and retained the transposon-encoded antibiotic resistance after incubation for three passages under nonselective condition. The frequency of auxotrophic mutants in the population confirmed that the insertion of the transposon was random and the C. freundii chromosome did not contain significant insertional hot spots of transposition. No extrachromosomal plasmid DNA was found. Conclusion Transposon mutagenesis of Mini-Tn5 is a suitable technique for the study of differentiation in C. freundii.
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Objective To reconstruct the new engineered bacteria expressing hPAB-? triploids so as to improve the outputs of recombinant human peptide antibiotic ?. Methods The recombinant plasmid pQE31-hPAB-?(3) was transformed into E. coli. M15 to screen the new engineered bacteria expressing hPAB-? triploids. The stabilities of phPAB-?(3)/M15 were observed in continuous cultures. The expression levels of the fusion peptides of interest and the bacterial yields of the new engineered bacteria phPAB-?(3)/M15 were compared with that of phPAB-?(3)/JM109 in different fermentation scales. Results Genetic stability of the recombinant plasmid and phPAB-?(3)/M15 was 100 after 10 passages. Take bacterial yields into account, the new engineered bacteria phPAB-?(3)/M15 was better than phPAB-?(3)/JM109 at the similar expression levels of the target proteins by “t” test analysis (P
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A strain of P. Aeruginosa,which was seperated from clinical environment ,shows a special characteristic . It keeps normal short rod shape when cultured at 37℃,however,it forms filament without pyocyanin producing when cultured at 25℃ overnight. The filaments will divide and form short rods,simultaneously,produce pyocyanin when culture time is prolonged to over 72h or culture temperature is raised to 37℃. The preliminary study indicates that this phenomenin has nothing to do with nutritive conditions and could the inbluenced by inoculating density and irradiating with ultraviolet rays The absence of pyocyanin was not the cause of filamentous formation by the test results.
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Objective To study the molecular mechanism of swarming motility in Citrobacter freundii.Methods Mini-Tn5 transposon was used to induce swarming mutants in Citrobacter freundii.The mutant genes were amplified by inverse-PCR and were then sequenced.Mutants were observed under light microscope and electron microscope to determine the characters of motility and bacterial shape.Results Seven swarming mutants were found to be three gene mutants(wzxB,waaW,waaL) involving LPS synthesis.Moreover,flagellar production of these mutants was abnormal,that is,only small portion of bacterial cells produced flagella normally.The majority of cells tended to aggregate and produced little flagella observed under electron microscope.Conclusion Absence of O-antigen affects the production of flagella and swarming motility of Citrobacter(freundii).