The gram-negative bacterium Acetobacter xylinum synthesizes an extracellular ribbon of cellulose microfibrils that possess unique structural and mechanical properties when compared to higher plant cellulose. All four...The gram-negative bacterium Acetobacter xylinum synthesizes an extracellular ribbon of cellulose microfibrils that possess unique structural and mechanical properties when compared to higher plant cellulose. All four genes in the cellulose-synthesizing operon (ayacs operon) of A. xylinum Ay201 were amplified by polymerase chain reaction (PCR) using oligonucleotide primers designed according to published acs operon sequence of A. xylinum ATCC 53582. Alignment of the two operons showed that they were highly homologous (98% similarity, 97% identity). AcsA and acsB gene were cloned in pCAMBIA 1301 vector while acsC and acsD were cloned in pCOB302-3 under the control of CaM 35S promoter. The constructs were introduced into cotton by the pollen-tube-pathway method and seeds obtained from putative transgenic plants were germinated on media containing hygromycin and phosphinothricin (PPT). Five seedlings out of 934 seeds were proved to contain all four foreign genes by PCR amplification. This is the first time that a whole operon encoding four different bacterial enzymes with various biological functions is transformed into cultivated cotton plants.展开更多
Background： o-galactosidase has been widely used in animal husbandry to reduce anti-nutritional factors （such as o-galactoside） in feed. Intestine-specific and substrate inducible expression of a-galactosidase woul...Background： o-galactosidase has been widely used in animal husbandry to reduce anti-nutritional factors （such as o-galactoside） in feed. Intestine-specific and substrate inducible expression of a-galactosidase would be highly beneficial for transgenic animal production. Methods： To achieve the intestine-specific and substrate inducible expression of o-galactosidase, we first identified intestine-specific promoters by comparing the transcriptional activity and tissue specificity of four intestine-specific promoters from human intestinal fatty acid binding protein, rat intestinal fatty acid binding protein, human mucin-2 and human lysozyme. We made two chimeric constructs combining the promoter and enhancer of human mucin-2, rat intestinal trefoil factor and human sucrase-isomaltase. Then a modified lac operon system was constructed to investigate the induction of o-galactosidase expression and enzyme activity by isopropyl p-D-]-thiogalactopyranoside （IPTG） and an a-galactosidase substrate, a-lactose. We declared that the research carried out on human （Zhai Yafeng） was in compliance with the Helsinki Declaration and experimental research on animals also followed internationally recognized guidelines. Results： The activity of the human mucin-2 promoter was about 2 to 3 times higher than that of other intestine-specific promoters. In the/ac operon system, the repressor significantly decreased （P 〈 0.05） luciferase activity by approximately 6.5-fold and reduced the percentage of cells expressing green fluorescent protein （GFP） by approximately 2-fold. In addition, the expression level of o-galactosidase mRNA was decreased by 6-fold and a-galactosidase activity was reduced by 8-fold. in line with our expectations, IPTG and a-lactose supplementation reversed （P 〈 O.O5） the inhibition and produced a 5-fold increase of luciferase activity, an 11-fold enhancement in the percentage of cells with GFP expression and an increase in o-galactosidase mfiNA abundance （by about 5-fold） and o-galactosidase activity （by about 7-fold）. Conclusions： We have successfully constructed a high specificity inducible lac operon system in an intestine-derived cell line, which could be of great value for gene therapy applications and transgenic animal production.展开更多
Fragment containing the whole riboflavin(rib)operons of B.cereus ATCC14579 was detected from GenBank and annotated.The rib operon of ATCC14579 was cloned with Pn,its native promoter,or with P43,the vegetative growth p...Fragment containing the whole riboflavin(rib)operons of B.cereus ATCC14579 was detected from GenBank and annotated.The rib operon of ATCC14579 was cloned with Pn,its native promoter,or with P43,the vegetative growth promoter,into the plasmid.Expression analysis showed that heterologous rib operon was operative in B.subtilis.Integrative plasmid with P43-rib fragment was integrated into the chromosome of B.subtilis RH33,yielding transformant B.subtilis PY.With optimized medium components,4.3 g·L -1 of riboflavin was achieved in batch culture of B.subtilis PY,which was 27%enhancement compared to the host strain.Real-time reverse transcription polymerase chain reaction(RT-PCR)analysis indicated that the transcriptional level of ribA maintained 2.8-fold higher with the expression of herterologous rib operon.Furthermore,the stability of B.subtilis PY was increased form 45%to 87%.The high transcriptional level of rib gene and higher stability of B.subtilis PY could explain the increased riboflavin production.展开更多
After deregulating the purine and riboflavin synthesis in the Gram-positive bacterium Bacillus subtilis,it is critical to amplify riboflavin operon with appropriate dosage in the host strain for remarkable increase of...After deregulating the purine and riboflavin synthesis in the Gram-positive bacterium Bacillus subtilis,it is critical to amplify riboflavin operon with appropriate dosage in the host strain for remarkable increase of riboflavin production.Bacillus subtilis RH13, a riboflavin-producing strain, was selected as host strain in the construction of engineering strains by protoplast fusion. The integrative plasmid pRB63 and autonomous plasmid pRB49, pRB62 containing riboflavin operon of B.subtilis 24 were constructed and transformed into the host strain respectively. Increasing one operon copy in B.subtilis RH13 results in about 0.4 g/L improvement in riboflavin yield and the appropriate number of operon copies was about 7—8. Amplifying more riboflavin operons is of no use for further improvement of yield of riboflavin. Furthermore, excessive operon dosage results in metabolic unbalance and is fatal to the host cells producing riboflavin.展开更多
Objective To understand the mechanism of invasion by Legionella dumoffii. Methods The L. dumoffii strain Tex-KL was mutated using the Tn903 derivative, Tn903 d IIlac Z. After screening 799 transposon insertion mutants...Objective To understand the mechanism of invasion by Legionella dumoffii. Methods The L. dumoffii strain Tex-KL was mutated using the Tn903 derivative, Tn903 d IIlac Z. After screening 799 transposon insertion mutants, we isolated one defective mutant. We then constructed the gene-disrupted mutant, KL16, and studied its invasion of and intracellular growth in He La and A549 cells, and in A/J mice survival experiments. The structure of traC-traD operon was analyzed by RT-PCR. Results The transposon insertion was in a gene homologous to Salmonella typhi tra C, which is required for the assembly of F pilin into the mature F pilus structure and for conjugal DNA transmission. Results from RT-PCR suggested that the traC-traD region formed an operon. We found that when the tra C gene was disrupted, invasion and intracellular growth of L. dumoffii Tex-KL were impaired in human epithelial cells. When mice were infected by intranasal inoculation with a tra C deficient mutant, their survival significantly increased when compared to mice infected with the wild-type strain. Conclusion Our results indicated that the traC-traD operon is required for the invasion and intracellular growth abilities of L. dumoffii Tex-KL in epithelial cells.展开更多
A riboflavin operon （rib operon） derived from Bacillus subtilis 368 was modified on structure and the resulting operons were expressed in various strains of Escherichia coli. The results showed that the optimization...A riboflavin operon （rib operon） derived from Bacillus subtilis 368 was modified on structure and the resulting operons were expressed in various strains of Escherichia coli. The results showed that the optimization of the rib operon and the host strain used for expression are two main factors affecting the riboflavin production. Replacing the promoterl and rfn box of the rib operon with a strong constructive promoter spol drastically increased the expression of the rib genes. When E. coli JM109 was used as the host strain, the highest riboflavin production reached 95.3μg/mL （about eight times higher than that of the unmodified r/b operon）. In addition, when tetracycline （20 μg/mL） was used as the selective pressure, compared with the ampicillin resistant transformants, a higher riboflavin yield was obtained in tetracycline resistant host strain.展开更多
Use of 16S-23S intergenic transcribed spacer (ITS) variability,as a relatively new method,is becoming an important supplement to the molecular methods based on 16S rRNA for which has a fairly constant size and is not ...Use of 16S-23S intergenic transcribed spacer (ITS) variability,as a relatively new method,is becoming an important supplement to the molecular methods based on 16S rRNA for which has a fairly constant size and is not divergent enough to give good separation in close relationships. This paper summarizes the structures and characteristics of ITS regions that are extremely variable in copy number,length and sequence per genome. The ITS region can be amplified easily taking advantage of conserved nucleotide stretches at the 5’ of the 16S and 3’ of the 23S gene,and the amplicon can contain different amounts of the 16S rDNA by choosing primers at different conserved areas within this gene. These primers are listed and discussed for perfecting the methodology of ITS. Furthermore,some recent progresses on the taxonomy,identification and community analysis of bacteria by means of ITS in epidemiology,ecology and artificial environment are reviewed,as well,the virtues and limitations of that method are discussed. Fig 2,Tab 1,Ref展开更多
针对宏-微机械手末端位姿、位置准确度的误差问题,受细胞学操纵子学说的启发,提出了基于操纵子模型的宏-微精密机械手运动的映射关系,研究精密机械手运动的影响因子和细胞学中的操纵子模型,分析机械手精密运动影响因子的影响方式和操纵...针对宏-微机械手末端位姿、位置准确度的误差问题,受细胞学操纵子学说的启发,提出了基于操纵子模型的宏-微精密机械手运动的映射关系,研究精密机械手运动的影响因子和细胞学中的操纵子模型,分析机械手精密运动影响因子的影响方式和操纵子模型的结构组成及其工作机理,结合操纵子模型的工作机理,分析宏-微机械手的控制系统,在此基础上,求解机械手精密运动控制系统的操纵子模型,考虑在几何因素的影响下,得出机械手末端位置在影响因子前后的误差值,通过基于粒子群算法的比例-积分-微分(Proportional-integral-differential,PID)控制器对误差结果进行优化,得出基于时间乘绝对误差积分准则(Time multiplied by the absolute error integral,ITAE)的粒子群算法(Particle swarm optimization,PSO)变化曲线和PID最优的控制参数。结论表明由映射关系推导出的控制系统能够满足一定的精度要求,并能进一步提高机械手的运动精度,为生物控制在机器人中进一步的创新研究提供了理论基础和依据。展开更多
文摘The gram-negative bacterium Acetobacter xylinum synthesizes an extracellular ribbon of cellulose microfibrils that possess unique structural and mechanical properties when compared to higher plant cellulose. All four genes in the cellulose-synthesizing operon (ayacs operon) of A. xylinum Ay201 were amplified by polymerase chain reaction (PCR) using oligonucleotide primers designed according to published acs operon sequence of A. xylinum ATCC 53582. Alignment of the two operons showed that they were highly homologous (98% similarity, 97% identity). AcsA and acsB gene were cloned in pCAMBIA 1301 vector while acsC and acsD were cloned in pCOB302-3 under the control of CaM 35S promoter. The constructs were introduced into cotton by the pollen-tube-pathway method and seeds obtained from putative transgenic plants were germinated on media containing hygromycin and phosphinothricin (PPT). Five seedlings out of 934 seeds were proved to contain all four foreign genes by PCR amplification. This is the first time that a whole operon encoding four different bacterial enzymes with various biological functions is transformed into cultivated cotton plants.
基金supported by the National Key Project (2009CB941601, 2008ZX08006-004 and 2009ZX0890-024B)the Joint Funds of the National Natural Science Foundation of China (u0731004)
文摘Background： o-galactosidase has been widely used in animal husbandry to reduce anti-nutritional factors （such as o-galactoside） in feed. Intestine-specific and substrate inducible expression of a-galactosidase would be highly beneficial for transgenic animal production. Methods： To achieve the intestine-specific and substrate inducible expression of o-galactosidase, we first identified intestine-specific promoters by comparing the transcriptional activity and tissue specificity of four intestine-specific promoters from human intestinal fatty acid binding protein, rat intestinal fatty acid binding protein, human mucin-2 and human lysozyme. We made two chimeric constructs combining the promoter and enhancer of human mucin-2, rat intestinal trefoil factor and human sucrase-isomaltase. Then a modified lac operon system was constructed to investigate the induction of o-galactosidase expression and enzyme activity by isopropyl p-D-]-thiogalactopyranoside （IPTG） and an a-galactosidase substrate, a-lactose. We declared that the research carried out on human （Zhai Yafeng） was in compliance with the Helsinki Declaration and experimental research on animals also followed internationally recognized guidelines. Results： The activity of the human mucin-2 promoter was about 2 to 3 times higher than that of other intestine-specific promoters. In the/ac operon system, the repressor significantly decreased （P 〈 0.05） luciferase activity by approximately 6.5-fold and reduced the percentage of cells expressing green fluorescent protein （GFP） by approximately 2-fold. In addition, the expression level of o-galactosidase mRNA was decreased by 6-fold and a-galactosidase activity was reduced by 8-fold. in line with our expectations, IPTG and a-lactose supplementation reversed （P 〈 O.O5） the inhibition and produced a 5-fold increase of luciferase activity, an 11-fold enhancement in the percentage of cells with GFP expression and an increase in o-galactosidase mfiNA abundance （by about 5-fold） and o-galactosidase activity （by about 7-fold）. Conclusions： We have successfully constructed a high specificity inducible lac operon system in an intestine-derived cell line, which could be of great value for gene therapy applications and transgenic animal production.
基金Supported by the National Natural Science Foundation of China（20536040） the State Key Development Program for Basic Research of China（2007CB707802） the Development Project of Science and Technology of Tianjin（05YFGZGX04500）
文摘Fragment containing the whole riboflavin(rib)operons of B.cereus ATCC14579 was detected from GenBank and annotated.The rib operon of ATCC14579 was cloned with Pn,its native promoter,or with P43,the vegetative growth promoter,into the plasmid.Expression analysis showed that heterologous rib operon was operative in B.subtilis.Integrative plasmid with P43-rib fragment was integrated into the chromosome of B.subtilis RH33,yielding transformant B.subtilis PY.With optimized medium components,4.3 g·L -1 of riboflavin was achieved in batch culture of B.subtilis PY,which was 27%enhancement compared to the host strain.Real-time reverse transcription polymerase chain reaction(RT-PCR)analysis indicated that the transcriptional level of ribA maintained 2.8-fold higher with the expression of herterologous rib operon.Furthermore,the stability of B.subtilis PY was increased form 45%to 87%.The high transcriptional level of rib gene and higher stability of B.subtilis PY could explain the increased riboflavin production.
文摘After deregulating the purine and riboflavin synthesis in the Gram-positive bacterium Bacillus subtilis,it is critical to amplify riboflavin operon with appropriate dosage in the host strain for remarkable increase of riboflavin production.Bacillus subtilis RH13, a riboflavin-producing strain, was selected as host strain in the construction of engineering strains by protoplast fusion. The integrative plasmid pRB63 and autonomous plasmid pRB49, pRB62 containing riboflavin operon of B.subtilis 24 were constructed and transformed into the host strain respectively. Increasing one operon copy in B.subtilis RH13 results in about 0.4 g/L improvement in riboflavin yield and the appropriate number of operon copies was about 7—8. Amplifying more riboflavin operons is of no use for further improvement of yield of riboflavin. Furthermore, excessive operon dosage results in metabolic unbalance and is fatal to the host cells producing riboflavin.
基金supported by the National Natural Scientific Foundation(No.81201251)from the Ministry of Science and Technology of the People’s Republic of Chinathe Priority Project on Infectious Disease Control and Prevention(No.2012ZX10004215 and 2013ZX10004-610-007)from the Ministry of Health and the Ministry of Science and Technology of the People’s Republic of Chinathe Science Foundation for the State Key Laboratory for Infectious Disease Prevention and Control from China(Grant No.2015SKLID508 and 2011SKLID202)
文摘Objective To understand the mechanism of invasion by Legionella dumoffii. Methods The L. dumoffii strain Tex-KL was mutated using the Tn903 derivative, Tn903 d IIlac Z. After screening 799 transposon insertion mutants, we isolated one defective mutant. We then constructed the gene-disrupted mutant, KL16, and studied its invasion of and intracellular growth in He La and A549 cells, and in A/J mice survival experiments. The structure of traC-traD operon was analyzed by RT-PCR. Results The transposon insertion was in a gene homologous to Salmonella typhi tra C, which is required for the assembly of F pilin into the mature F pilus structure and for conjugal DNA transmission. Results from RT-PCR suggested that the traC-traD region formed an operon. We found that when the tra C gene was disrupted, invasion and intracellular growth of L. dumoffii Tex-KL were impaired in human epithelial cells. When mice were infected by intranasal inoculation with a tra C deficient mutant, their survival significantly increased when compared to mice infected with the wild-type strain. Conclusion Our results indicated that the traC-traD operon is required for the invasion and intracellular growth abilities of L. dumoffii Tex-KL in epithelial cells.
文摘A riboflavin operon （rib operon） derived from Bacillus subtilis 368 was modified on structure and the resulting operons were expressed in various strains of Escherichia coli. The results showed that the optimization of the rib operon and the host strain used for expression are two main factors affecting the riboflavin production. Replacing the promoterl and rfn box of the rib operon with a strong constructive promoter spol drastically increased the expression of the rib genes. When E. coli JM109 was used as the host strain, the highest riboflavin production reached 95.3μg/mL （about eight times higher than that of the unmodified r/b operon）. In addition, when tetracycline （20 μg/mL） was used as the selective pressure, compared with the ampicillin resistant transformants, a higher riboflavin yield was obtained in tetracycline resistant host strain.
文摘Use of 16S-23S intergenic transcribed spacer (ITS) variability,as a relatively new method,is becoming an important supplement to the molecular methods based on 16S rRNA for which has a fairly constant size and is not divergent enough to give good separation in close relationships. This paper summarizes the structures and characteristics of ITS regions that are extremely variable in copy number,length and sequence per genome. The ITS region can be amplified easily taking advantage of conserved nucleotide stretches at the 5’ of the 16S and 3’ of the 23S gene,and the amplicon can contain different amounts of the 16S rDNA by choosing primers at different conserved areas within this gene. These primers are listed and discussed for perfecting the methodology of ITS. Furthermore,some recent progresses on the taxonomy,identification and community analysis of bacteria by means of ITS in epidemiology,ecology and artificial environment are reviewed,as well,the virtues and limitations of that method are discussed. Fig 2,Tab 1,Ref
文摘针对宏-微机械手末端位姿、位置准确度的误差问题,受细胞学操纵子学说的启发,提出了基于操纵子模型的宏-微精密机械手运动的映射关系,研究精密机械手运动的影响因子和细胞学中的操纵子模型,分析机械手精密运动影响因子的影响方式和操纵子模型的结构组成及其工作机理,结合操纵子模型的工作机理,分析宏-微机械手的控制系统,在此基础上,求解机械手精密运动控制系统的操纵子模型,考虑在几何因素的影响下,得出机械手末端位置在影响因子前后的误差值,通过基于粒子群算法的比例-积分-微分(Proportional-integral-differential,PID)控制器对误差结果进行优化,得出基于时间乘绝对误差积分准则(Time multiplied by the absolute error integral,ITAE)的粒子群算法(Particle swarm optimization,PSO)变化曲线和PID最优的控制参数。结论表明由映射关系推导出的控制系统能够满足一定的精度要求,并能进一步提高机械手的运动精度,为生物控制在机器人中进一步的创新研究提供了理论基础和依据。
