Construction of T7 RNA polymerase gene expression system in Anabaena sp. PCC 7120 for the expression of hG-CSF / 生物工程学报

The low expression rate of exogenous genes in cyanobacteria is one of the bottlenecks of cyanobacteria genetic engineering. The T7 RNA polymerase expression system has achieved the efficient expression of exogenous genes in Escherichia coli. Cyanobacteria and E. coli are both Gram-negative bacteria with high genetic homology. The construction of T7 RNA polymerase expression system in cyanobacteria may improve the expression of foreign genes. In order to construct the T7 RNA polymerase expression system in Anabaena sp. PCC 7120, methods such as overlapping extension PCR and digestion-ligation technique were used to construct a site-specific integration vector pEASY-T1-F1-TacT7RNAPCmR-F2 and a shuttle expression vector pRL-T7-hG-CSF. The site-specific integration vector is capable of expressing T7 RNA polymerase, and the shuttle expression vector expresses hG-CSF driven by the T7 promoter. Then we introduced the site-specific integration vector into the wild type cyanobacteria by electroporation and transferred the shuttle expression vector into the site-integrated transgenic cyanobacteria by triparental conjugative transfer. In the end, we identified the presence of foreign genes in cyanobacteria by PCR, tested the transcription level of foreign genes in cyanobacteria by RT-PCR, and detected the protein expression of foreign genes in cyanobacteria by Western blotting. The two vectors were successfully constructed, the T7 RNA polymerase gene and hG-CSF gene were transferred into cyanobacteria well, and both genes were also expressed in cyanobacteria. In summary, the T7 RNA polymerase expression system was successfully constructed in cyanobacteria, and the expression rate of hG-CSF gene was doubled than the traditional cyanobacteria expression systems. This expression system will provide a better tool for the application of cyanobacteria genetic engineering and will promote the development of cyanobacteria as a chassis cell in the fields of synthetic biology in the future.

Sequence analysis of plasmid in Klebsiella pneumoniae KF3 / 中华微生物学和免疫学杂志

Objective To study the structures of the plasmids of Klebsiella pneumoniae KF3 at the genome metagenome level througth with whole plasmid DNA sequencing, to analyze the functional genes carried by plasmid and to identify the correlation of resistance and pathogenicity between the plasmids and the host strains. Methods The alkaline lysis method was used to extract plasmids. We constructed the small insert pUC18 library and the large insert Forsmid library, sequenced and used the Phred / Phrap / Consed package to assemble these sequences and gained a complete sequence. The open reading frame(ORFs) were predicted by the Glimmer software and annotated, analyzed the functions of these genes. Results We successfully constructed the pUC18 library and the Fosmid libraries for the plasmid DNA and obtained three circular double-stranded DNA plasmids: pKF3-70 (69 477 bp), pKF3-90 (91 327 bp) and pKF3-147 ( 147 416 bp). There were drug resistant genes, conjugative transfer genes and mobile DNA elements identified on three plasmids. Conclusion The three plasmids of KF3 could be transferred among different strains. It would lead to the dissemination of the resistant genes.

The Mechanism of Al_2O_3 Nanoparticle Influence on RP4-mediated Conjugation:a Preliminary Research in Bacterium Morphology / 环境与健康杂志

Objective To explore the influence of Al2O3 nanoparticle (20nm in average) on RP4-mediated conjugation and its mechanism was explored further. Methods Mating was conducted between E.coli HB101(RP4) and Salmonella aberdeen Kauffmann 50312(strR) in saline at 25 ℃ without stirring for 8 h,the concentrations of Al2O3 nanoparticle were 0.005,0.05,0.5,5,50 mmol/L respectively. The initial concentration of donor and recipient were both about 109 cfu/ml(donor per recipient ratio was 1∶3).Later transmission electron microscope(TEM) and laser scan confocal microscope (LSCM) were exploited to investigate cell morphology and structure. Results 5 mmol/L and 50 mmol/L Al2O3 nanoparticle could increase the conjugal transfer frequency by 150-fold and 40-fold respectively. TEM observation on thin section indicated several bacterial were prone to form conjugational junction together in 5 mmol/L and 50 mmol/L Al2O3 nanoparticle treated group,which was rarely seen in control group. Meanwhile the cell envelope of bacterial was significantly damaged upon the treatment by 50 mmol/L Al2O3,which might be the reason why transfer frequency of 50 mmol/L Al2O3 group was less than 5 mmol/L Al2O3 group. LSCM result indicated that Al2O3 nanoparticle might damage cell envelope and the damage was positively related to the accumulation of Al2O3 in bacterial. Conclusion Al2O3 nanoparticle can stimulate RP4 conjugal transfer. Conjugative gene is highly regulated. Al2O3 nanoparticle might modulate conjugative gene expression directly or influence cell membrane permeability and thus modulate the process of conjugation indirectly .