Objective:To identify the specific integration site of prophage φ297 in the host of E. coli K12 chromosome. Methods:Using molecular techniques such as Siebert PCR for walking from the int gene of prophage 297, which ...Objective:To identify the specific integration site of prophage φ297 in the host of E. coli K12 chromosome. Methods:Using molecular techniques such as Siebert PCR for walking from the int gene of prophage 297, which is similar to that of phage 933W to an unknown region in genomic DNA. A special adaptor is ligated to the ends of DNA fragments generated by digestion of genomic DNA with restriction enzymes that generates blunt ended fragments. Clone and subclone of PCR products, DNA sequencing and data analysis were used in this study. Results:The attL, attR and the core sequences were determined. The bacterial attachment site of phage φ297 was located in the yecE gene of E. coli K12. Conclusion:The phage φ297 integrates into the yecE gene of the E. coli K12 genome.展开更多
Lacto-N-neotetraose(LNnT),one of the most important human milk oligosaccharides,can be used as infants’food addi-tives.Nowadays,extraction,chemical and biological synthesis were utilized to obtain LNnT,while these me...Lacto-N-neotetraose(LNnT),one of the most important human milk oligosaccharides,can be used as infants’food addi-tives.Nowadays,extraction,chemical and biological synthesis were utilized to obtain LNnT,while these methods still face some problems such as low yield and high cost.The aim of current work is to construct a de novo biosynthesis pathway of LNnT in E.coli K12 MG1655.The lgtA and lgtB were first expressed by a plasmid,resulting in a LNnT titer of 0.04 g/L.To improve the yield of LNnT on substrate lactose,lacZ and lacI were knocked out,and lacY was over-expressed.As a result,the yield of LNnT on lactose increased from 0.01 to 0.09 mol/mol,and the titer of LNnT elevated to 0.41 g/L.In addition,the pathway was regulated using the titer of Lacto-N-triose II(LNTII)as a measure,and obtained a high titer strain of LNnT for 1.04 g/L.Finally,the gene expressions were fine-tuned,the titer of LNnT reached 1.2 g/L,which was 93%higher than the control strain,and the yield on lactose reached 0.28 mol/mol.The engineering strategy of pathway construction and modulation used in this study is applicable to facilitate the microbial production of other metabolites in E.coli.展开更多
Comparative genome analysis is performed between Shigella flexneri 2a strain 301 and its close relatives, the nonpathogenic E. Coli K-12 strain MG1655. Result shows that there are 136 DNA segments whose size is larger...Comparative genome analysis is performed between Shigella flexneri 2a strain 301 and its close relatives, the nonpathogenic E. Coli K-12 strain MG1655. Result shows that there are 136 DNA segments whose size is larger than 1000 bp absent from Shigella flexneri 2a strain 301, which is up to 717253 bp in total length. These deleted segments altogether contain 670 open reading frames (ORFs). Prediction of these ORFs indicates that there are 40% genes of unknown function. The other genes of definite functions encode metabolic enzymes, structure proteins, transcription regulatory factors and some elements correlated with horizontal transfer. Here we compare the complete genomic sequences of the two closely related species, which differ in pathogenic phenotype. To our knowledge, this not only reveals the difference of genomic sequence between the two important enteric pathogens for the first time, but also provides valuable clues to further researches in its process of physiological activity, pathogenesis and the evolution of enteric bacteria.展开更多
The three parts(Stx17B, Stx27B and StxB) of Shiga toxin B subunit have been fused into a cell surface exposed loop of the LamB protein at a BamH I site between residues 153 and 154. Western blotting revealed that the ...The three parts(Stx17B, Stx27B and StxB) of Shiga toxin B subunit have been fused into a cell surface exposed loop of the LamB protein at a BamH I site between residues 153 and 154. Western blotting revealed that the three parts of Shiga toxin B subunit could be expressed as the Lamb fusion proteins in E. coli. Indirect immunofluorescence and immunoelectron microscopy analyses showed fusion proteins LamB/Stx17B and LamB/Stx27B could be expressed at cell surface in E. coli, but fusion protein LamB/StxB could not be expressed at cell surface; it was aggregated in cytoplasm and was toxic to host. This expression system provided a new way to construct an oral live vaccine against Shigella dysenteriae 1.展开更多
文摘Objective:To identify the specific integration site of prophage φ297 in the host of E. coli K12 chromosome. Methods:Using molecular techniques such as Siebert PCR for walking from the int gene of prophage 297, which is similar to that of phage 933W to an unknown region in genomic DNA. A special adaptor is ligated to the ends of DNA fragments generated by digestion of genomic DNA with restriction enzymes that generates blunt ended fragments. Clone and subclone of PCR products, DNA sequencing and data analysis were used in this study. Results:The attL, attR and the core sequences were determined. The bacterial attachment site of phage φ297 was located in the yecE gene of E. coli K12. Conclusion:The phage φ297 integrates into the yecE gene of the E. coli K12 genome.
基金This work was supported by the National Natural Science Foundation of China(31930085,32021005)the key research and development program of China(2018YFA0900300,2020YFA0908300).
文摘Lacto-N-neotetraose(LNnT),one of the most important human milk oligosaccharides,can be used as infants’food addi-tives.Nowadays,extraction,chemical and biological synthesis were utilized to obtain LNnT,while these methods still face some problems such as low yield and high cost.The aim of current work is to construct a de novo biosynthesis pathway of LNnT in E.coli K12 MG1655.The lgtA and lgtB were first expressed by a plasmid,resulting in a LNnT titer of 0.04 g/L.To improve the yield of LNnT on substrate lactose,lacZ and lacI were knocked out,and lacY was over-expressed.As a result,the yield of LNnT on lactose increased from 0.01 to 0.09 mol/mol,and the titer of LNnT elevated to 0.41 g/L.In addition,the pathway was regulated using the titer of Lacto-N-triose II(LNTII)as a measure,and obtained a high titer strain of LNnT for 1.04 g/L.Finally,the gene expressions were fine-tuned,the titer of LNnT reached 1.2 g/L,which was 93%higher than the control strain,and the yield on lactose reached 0.28 mol/mol.The engineering strategy of pathway construction and modulation used in this study is applicable to facilitate the microbial production of other metabolites in E.coli.
基金supported by the State“973”Key Basic Research Program(Grant No.G1999054103)the State“863”High-Tech Project(Grant No.Z19-02-05-01)+1 种基金Beijing Innovation Engineering(Grant No.955020700)the North China Pharmaceutical Corporation(NCPC)
文摘Comparative genome analysis is performed between Shigella flexneri 2a strain 301 and its close relatives, the nonpathogenic E. Coli K-12 strain MG1655. Result shows that there are 136 DNA segments whose size is larger than 1000 bp absent from Shigella flexneri 2a strain 301, which is up to 717253 bp in total length. These deleted segments altogether contain 670 open reading frames (ORFs). Prediction of these ORFs indicates that there are 40% genes of unknown function. The other genes of definite functions encode metabolic enzymes, structure proteins, transcription regulatory factors and some elements correlated with horizontal transfer. Here we compare the complete genomic sequences of the two closely related species, which differ in pathogenic phenotype. To our knowledge, this not only reveals the difference of genomic sequence between the two important enteric pathogens for the first time, but also provides valuable clues to further researches in its process of physiological activity, pathogenesis and the evolution of enteric bacteria.
文摘The three parts(Stx17B, Stx27B and StxB) of Shiga toxin B subunit have been fused into a cell surface exposed loop of the LamB protein at a BamH I site between residues 153 and 154. Western blotting revealed that the three parts of Shiga toxin B subunit could be expressed as the Lamb fusion proteins in E. coli. Indirect immunofluorescence and immunoelectron microscopy analyses showed fusion proteins LamB/Stx17B and LamB/Stx27B could be expressed at cell surface in E. coli, but fusion protein LamB/StxB could not be expressed at cell surface; it was aggregated in cytoplasm and was toxic to host. This expression system provided a new way to construct an oral live vaccine against Shigella dysenteriae 1.
