Objective: To identify the region conferring stability to pBSSB2(a linear plasmid, pBSSB1, containing a kanamycin cassette), which is unique to Indonesian isolates of Salmonella enterica serovar Typhi. Methods: The op...Objective: To identify the region conferring stability to pBSSB2(a linear plasmid, pBSSB1, containing a kanamycin cassette), which is unique to Indonesian isolates of Salmonella enterica serovar Typhi. Methods: The open reading frame(ORF) 009 was identified as a toxin coding gene in the plasmid through introduction of translational termination codons in the ORF. Results: The stability function was located in a fragment that spanned nucleotides 5 766 to 6 828 in the linear plasmid genetic map. Ectopic expression of ORF009 in pBAD18 vector indicated ORF009 codes for a toxin. This fragment could stabilize plasmid pUC18 previously destabilized through mutation of the pcnB(plasmid copy number control) gene that codes for polyA polymerase. Majority of the cells expressing ORF009 were non-viable according to phase contrast microscopy. Conclusions: This study demonstrated that a linear plasmid fragment that carries a gene encoding a toxin possibly conferred stability to the parent plasmid. It was able to stabilize a multicopy plasmid of Escherichia coli.展开更多
Bacillus thuringiensis,a safe bacterium widely used in agriculture for the biocontrol of pests,has great potential for protein production.The linear plasmid expression system,bacterial orthogonal DNA replication syste...Bacillus thuringiensis,a safe bacterium widely used in agriculture for the biocontrol of pests,has great potential for protein production.The linear plasmid expression system,bacterial orthogonal DNA replication system constructed based on B.thuringiensis prophage GIL16,can achieve stable and high levels of gene expression in the absence of external selection pressure,facilitating development of B.thuringiensis chassis cells.However,the regulatory elements of gene expression and protein secretion suitable for the B.thuringiensis expression system are still lacking.Therefore,the development and optimization of different genetic tools are required.We constructed a promoter library containing 107 different-strength promoters(covering persistently high/intermediate/low level)by transcriptomic analysis of the cell at different growth stages and a signal peptide library(59 signal peptides from Bacillus subtilis and four endogenous signal peptides from B.thuringiensis)to enrich the genetic toolbox using alpha-lactalbumin(α-LA)as the characterization product.Then,a high-throughput microfluidic screening platform based on BacORep and self-assembled split fluorescent protein was developed to further optimize expression elements,resulting in an improved α-LA-producing B.thuringiensis.Finally,the maximum copy number of linear plasmids was 9.3 times higher than that of the original.The titer of α-LA reached 107.7 mg/L in a 3 L bioreactor,which was comparable to the highest yield reported in Komagataella phaffii.We substantially expanded the synthetic biology toolbox for linear plasmid expression systems and provided a strategy for creating efficient prokaryotic expression system.展开更多
Four kinds of mitochondrial plasmid-like DNAs, designated pC1, pC2, pC3 and pC4, were detected in Cucumis sativus Jinyan No. 4. The electron microscopy observation showed that pC4 was linear conformation. Complete seq...Four kinds of mitochondrial plasmid-like DNAs, designated pC1, pC2, pC3 and pC4, were detected in Cucumis sativus Jinyan No. 4. The electron microscopy observation showed that pC4 was linear conformation. Complete sequence of pC4 was cloned into pUC19 with E. coli JM109 as host. Sequence analysis revealed that pC4 was 370 bp long, the shortest one among all the reported mitochondrial plasmid-like DNAs. pC4 was AT rich. It contained terminal direct repeat sequence (35 bp in length)as well as many short direct and inverted repeats. ORFs in pC4 were short. pC4 was found to be homologous to nuclear DNAs, but lack homology with main mitochondrial and chloroplast DNAs. pC4-homologous sequence also occurred in nuclear genome of Jinyan No. 7 which contained no mitochondrial plasmid-like DNAs. The hybridization pattern of Jinyan No. 7 was slightly different from that of Jinyan No. 4. This suggested that pC4 occurred at the forepart of Cucumis sativus species divergence and integrated into the nuclear genome,展开更多
Using pulsed\|field gel electrophoresis (PFGE),two indigenous large plasmids were isolated from Streptomyces T8 4.Two dimentional PFGE revealed that both plasmids were linear molecules.By parallel electrophoresis with...Using pulsed\|field gel electrophoresis (PFGE),two indigenous large plasmids were isolated from Streptomyces T8 4.Two dimentional PFGE revealed that both plasmids were linear molecules.By parallel electrophoresis with linear plasmids of known sizes,the two linear plasmids were estimated to be approximately 230kb and 90kb,which were designated as pHZ1000 and pHZ1001,respectively.Both plasmids could be transferred into S.lividans ZX1 by conjugation,which is detectable by “pock”formation.Five S.lividans ZX1 derivatives which carry one or two plasmids were isolated and characterized by Southern hybridization and PFGE.展开更多
Bacterial chromosomes and plasmids are commonly circular, however, lin ear chromosomes and plasmids were discovered among 5 genera of the Actinomycet ales. Here, we use pulsed field gel electrophoresis to study the ge...Bacterial chromosomes and plasmids are commonly circular, however, lin ear chromosomes and plasmids were discovered among 5 genera of the Actinomycet ales. Here, we use pulsed field gel electrophoresis to study the genomes of 19 spe cies which belong to 15 genera in the Actinomycetales. All chromosomes of 19 spe cies are linear DNA, and linear plasmids with different sizes and copy numbers a re detected among 5 species. This work provide basis for investigating the possi ble novel functions of linear replicons beyond Streptomyces and also helps t o develop Actinomycetales artificial linear chromosome.展开更多
文摘Objective: To identify the region conferring stability to pBSSB2(a linear plasmid, pBSSB1, containing a kanamycin cassette), which is unique to Indonesian isolates of Salmonella enterica serovar Typhi. Methods: The open reading frame(ORF) 009 was identified as a toxin coding gene in the plasmid through introduction of translational termination codons in the ORF. Results: The stability function was located in a fragment that spanned nucleotides 5 766 to 6 828 in the linear plasmid genetic map. Ectopic expression of ORF009 in pBAD18 vector indicated ORF009 codes for a toxin. This fragment could stabilize plasmid pUC18 previously destabilized through mutation of the pcnB(plasmid copy number control) gene that codes for polyA polymerase. Majority of the cells expressing ORF009 were non-viable according to phase contrast microscopy. Conclusions: This study demonstrated that a linear plasmid fragment that carries a gene encoding a toxin possibly conferred stability to the parent plasmid. It was able to stabilize a multicopy plasmid of Escherichia coli.
基金financially supported by the National Science Fund for Excellent Young Scholars(32222069)National Natural Science Foundation of China(32172349)+1 种基金Foundation for Innovative Research Groups of the National Natural Science Foundation of China(32021005)National Key Research and Development Program of China(2020YFA0908300).
文摘Bacillus thuringiensis,a safe bacterium widely used in agriculture for the biocontrol of pests,has great potential for protein production.The linear plasmid expression system,bacterial orthogonal DNA replication system constructed based on B.thuringiensis prophage GIL16,can achieve stable and high levels of gene expression in the absence of external selection pressure,facilitating development of B.thuringiensis chassis cells.However,the regulatory elements of gene expression and protein secretion suitable for the B.thuringiensis expression system are still lacking.Therefore,the development and optimization of different genetic tools are required.We constructed a promoter library containing 107 different-strength promoters(covering persistently high/intermediate/low level)by transcriptomic analysis of the cell at different growth stages and a signal peptide library(59 signal peptides from Bacillus subtilis and four endogenous signal peptides from B.thuringiensis)to enrich the genetic toolbox using alpha-lactalbumin(α-LA)as the characterization product.Then,a high-throughput microfluidic screening platform based on BacORep and self-assembled split fluorescent protein was developed to further optimize expression elements,resulting in an improved α-LA-producing B.thuringiensis.Finally,the maximum copy number of linear plasmids was 9.3 times higher than that of the original.The titer of α-LA reached 107.7 mg/L in a 3 L bioreactor,which was comparable to the highest yield reported in Komagataella phaffii.We substantially expanded the synthetic biology toolbox for linear plasmid expression systems and provided a strategy for creating efficient prokaryotic expression system.
基金This work was supported by the National Natural Science Foundation of China (Grant No. 39770414).
文摘Four kinds of mitochondrial plasmid-like DNAs, designated pC1, pC2, pC3 and pC4, were detected in Cucumis sativus Jinyan No. 4. The electron microscopy observation showed that pC4 was linear conformation. Complete sequence of pC4 was cloned into pUC19 with E. coli JM109 as host. Sequence analysis revealed that pC4 was 370 bp long, the shortest one among all the reported mitochondrial plasmid-like DNAs. pC4 was AT rich. It contained terminal direct repeat sequence (35 bp in length)as well as many short direct and inverted repeats. ORFs in pC4 were short. pC4 was found to be homologous to nuclear DNAs, but lack homology with main mitochondrial and chloroplast DNAs. pC4-homologous sequence also occurred in nuclear genome of Jinyan No. 7 which contained no mitochondrial plasmid-like DNAs. The hybridization pattern of Jinyan No. 7 was slightly different from that of Jinyan No. 4. This suggested that pC4 occurred at the forepart of Cucumis sativus species divergence and integrated into the nuclear genome,
文摘Using pulsed\|field gel electrophoresis (PFGE),two indigenous large plasmids were isolated from Streptomyces T8 4.Two dimentional PFGE revealed that both plasmids were linear molecules.By parallel electrophoresis with linear plasmids of known sizes,the two linear plasmids were estimated to be approximately 230kb and 90kb,which were designated as pHZ1000 and pHZ1001,respectively.Both plasmids could be transferred into S.lividans ZX1 by conjugation,which is detectable by “pock”formation.Five S.lividans ZX1 derivatives which carry one or two plasmids were isolated and characterized by Southern hybridization and PFGE.
文摘Bacterial chromosomes and plasmids are commonly circular, however, lin ear chromosomes and plasmids were discovered among 5 genera of the Actinomycet ales. Here, we use pulsed field gel electrophoresis to study the genomes of 19 spe cies which belong to 15 genera in the Actinomycetales. All chromosomes of 19 spe cies are linear DNA, and linear plasmids with different sizes and copy numbers a re detected among 5 species. This work provide basis for investigating the possi ble novel functions of linear replicons beyond Streptomyces and also helps t o develop Actinomycetales artificial linear chromosome.
