In order to establish a possible evolutionary correlation between α lactalbumin and lysozyme,site\|directed mutagenesis on α\|lactalbumin cDNA was performed to change α\|lactalbumin into lysozyme.The gene of α\|la...In order to establish a possible evolutionary correlation between α lactalbumin and lysozyme,site\|directed mutagenesis on α\|lactalbumin cDNA was performed to change α\|lactalbumin into lysozyme.The gene of α\|lactalbumin was cloned into phagemid pSK +vector and the single strand template of which was prepared.Thereafter,the mutagenic oligonucleotide primers were designed and used to synthesize the mutated double strand DNA.By means of this site\|directed mutagenesis,the amimo acid residues at position His 32 and Thr 33 of bovine α\|lactalbumin cDNA were substituted with Leu and Glu,respectively.Thus,the catalytic site of lysozyme was obtained in α\|lactalbumin.Furthermore,Tyr 103 was substituted by Ala and the lysozyme substrate binding conformation was formed in α\|lactalbumin as well.The structural as well as functional relationship between α\|lactalbumin and lysozyme indicated that there existed possible evolutionary correlation between the two proteins.展开更多
Site_specific mutagenesis has been widely used in molecular biology and biochemistry. The authors have developed a simple and easy method for site_specific mutagenesis of any genes on plasmids using long distance inve...Site_specific mutagenesis has been widely used in molecular biology and biochemistry. The authors have developed a simple and easy method for site_specific mutagenesis of any genes on plasmids using long distance inverse PCR in the presence of Pfu_DNA polymerase. The efficiency of this method is higher than 90% and the entire procedure can be performed just in one tube. No subcloning is needed. This method is especially useful for obtaining mutant genes on large plasmids such as Ti plasmids used for plant transformation.展开更多
With the availability of the whole genome sequence of Escherichia coli or Corynebacterium glutamicum, strategies for directed DNA manipulation have developed rapidly. DNA manipulation plays an important role in unders...With the availability of the whole genome sequence of Escherichia coli or Corynebacterium glutamicum, strategies for directed DNA manipulation have developed rapidly. DNA manipulation plays an important role in understanding the function of genes and in constructing novel engineering bacteria according to requirement. DNA manipulation involves modifying the autologous genes and expressing the heterogenous genes. Two alternative approaches, using electroporation linear DNA or recombinant suicide plasmid, allow a wide variety of DNA manipulation. However, the over-expression of the desired gene is generally executed via plasmid-mediation. The current review summarizes the common strategies used for genetically modifying E. coli and C. glutamicum genomes, and discusses the technical problem of multi-layered DNA manipulation. Strategies for gene over-expression via integrating into genome are proposed. This review is intended to be an accessible introduction to DNA manipulation within the bacterial genome for novices and a source of the latest experimental information for experienced investigators.展开更多
文摘In order to establish a possible evolutionary correlation between α lactalbumin and lysozyme,site\|directed mutagenesis on α\|lactalbumin cDNA was performed to change α\|lactalbumin into lysozyme.The gene of α\|lactalbumin was cloned into phagemid pSK +vector and the single strand template of which was prepared.Thereafter,the mutagenic oligonucleotide primers were designed and used to synthesize the mutated double strand DNA.By means of this site\|directed mutagenesis,the amimo acid residues at position His 32 and Thr 33 of bovine α\|lactalbumin cDNA were substituted with Leu and Glu,respectively.Thus,the catalytic site of lysozyme was obtained in α\|lactalbumin.Furthermore,Tyr 103 was substituted by Ala and the lysozyme substrate binding conformation was formed in α\|lactalbumin as well.The structural as well as functional relationship between α\|lactalbumin and lysozyme indicated that there existed possible evolutionary correlation between the two proteins.
文摘Site_specific mutagenesis has been widely used in molecular biology and biochemistry. The authors have developed a simple and easy method for site_specific mutagenesis of any genes on plasmids using long distance inverse PCR in the presence of Pfu_DNA polymerase. The efficiency of this method is higher than 90% and the entire procedure can be performed just in one tube. No subcloning is needed. This method is especially useful for obtaining mutant genes on large plasmids such as Ti plasmids used for plant transformation.
基金supported by the Natural Science Foundation of Jiangsu Province(No.BK20150149)the Fundamental Research Funds for the Central Universities(No.JUSRP51504)the Youth Foundation of Jiangnan University(No.JUSRP115A19),China
文摘With the availability of the whole genome sequence of Escherichia coli or Corynebacterium glutamicum, strategies for directed DNA manipulation have developed rapidly. DNA manipulation plays an important role in understanding the function of genes and in constructing novel engineering bacteria according to requirement. DNA manipulation involves modifying the autologous genes and expressing the heterogenous genes. Two alternative approaches, using electroporation linear DNA or recombinant suicide plasmid, allow a wide variety of DNA manipulation. However, the over-expression of the desired gene is generally executed via plasmid-mediation. The current review summarizes the common strategies used for genetically modifying E. coli and C. glutamicum genomes, and discusses the technical problem of multi-layered DNA manipulation. Strategies for gene over-expression via integrating into genome are proposed. This review is intended to be an accessible introduction to DNA manipulation within the bacterial genome for novices and a source of the latest experimental information for experienced investigators.
