A plasmid (pTU9) containing the lambda (λ) phage lysis genes S( )RRz and the biosynthetic genes phb CAB of poly β hydroxybutyrate (PHB) was constructed and transformed into E.coli JM109. Cultured in Luria Ber...A plasmid (pTU9) containing the lambda (λ) phage lysis genes S( )RRz and the biosynthetic genes phb CAB of poly β hydroxybutyrate (PHB) was constructed and transformed into E.coli JM109. Cultured in Luria Bertani (LB) medium with 20 g/L glucose, E.coli JM109 (pTU9) could accumulate PHB in cells up to 40% (g PHB per g dry cells). A chelating agent EDTA was applied to induce a complete cell lysis and PHB granules were released. This method has a potential application in PHB separation.展开更多
目的构建人肺癌细胞A549λ噬菌体cDNA文库,为抗肺癌药物的筛选和药物作用分子机理的研究奠定基础。方法提取人肺癌细胞A549λ总RNA,用DnaseⅠ处理后,采用RNA 5’末端移动反转录(switching mechanism at 5′end of RNA transcript,SMART...目的构建人肺癌细胞A549λ噬菌体cDNA文库,为抗肺癌药物的筛选和药物作用分子机理的研究奠定基础。方法提取人肺癌细胞A549λ总RNA,用DnaseⅠ处理后,采用RNA 5’末端移动反转录(switching mechanism at 5′end of RNA transcript,SMART)技术合成双链cDNA,切胶回收去除短片段,依次用蛋白酶K和SfiⅠ进行酶切,并与经过酶切的λTriplEx2载体连接后进行体外包装,得到初级文库。初级文库扩增后,测定扩增文库滴度,并通过PCR鉴定插入片段。结果构建的人肺癌细胞A549λ噬菌体初级文库库容为1.88×10^(7)pfu/mL,扩增文库滴度为2.50×10^(9)pfu/mL,插入cDNA片段为500~3000 bp。结论成功构建了人肺癌细胞A549λ噬菌体cDNA文库,为进一步筛选治疗肺癌的药物作用分子机理奠定了基础。展开更多
文摘A plasmid (pTU9) containing the lambda (λ) phage lysis genes S( )RRz and the biosynthetic genes phb CAB of poly β hydroxybutyrate (PHB) was constructed and transformed into E.coli JM109. Cultured in Luria Bertani (LB) medium with 20 g/L glucose, E.coli JM109 (pTU9) could accumulate PHB in cells up to 40% (g PHB per g dry cells). A chelating agent EDTA was applied to induce a complete cell lysis and PHB granules were released. This method has a potential application in PHB separation.
文摘目的构建人肺癌细胞A549λ噬菌体cDNA文库,为抗肺癌药物的筛选和药物作用分子机理的研究奠定基础。方法提取人肺癌细胞A549λ总RNA,用DnaseⅠ处理后,采用RNA 5’末端移动反转录(switching mechanism at 5′end of RNA transcript,SMART)技术合成双链cDNA,切胶回收去除短片段,依次用蛋白酶K和SfiⅠ进行酶切,并与经过酶切的λTriplEx2载体连接后进行体外包装,得到初级文库。初级文库扩增后,测定扩增文库滴度,并通过PCR鉴定插入片段。结果构建的人肺癌细胞A549λ噬菌体初级文库库容为1.88×10^(7)pfu/mL,扩增文库滴度为2.50×10^(9)pfu/mL,插入cDNA片段为500~3000 bp。结论成功构建了人肺癌细胞A549λ噬菌体cDNA文库,为进一步筛选治疗肺癌的药物作用分子机理奠定了基础。
