【摘要】 应用一种简单的方法构建出一株大肠杆菌脂肪酸降解基因B(fadB)缺陷型菌株KM32B(fadB∷Tet).该方法不需克隆和第二轮PCR,只需要一对各70个碱基的引物.引物的前45个碱基分别与大肠杆菌目的基因的3′和5′互补,后25个碱基分别与抗性(四环素Tet)基因的3′和5′互补.将PCR产物,即抗性基因两侧各带与目的基因3′和5′互补的45个碱基的片段,通过高频电转化入大肠杆菌KM32菌株.重组子经PCR验证整个fadB基因区域因被四环素基因置换而敲除.将两种BurkholderiacaryophylliAS1.2741聚羟基脂肪酸酯合成酶基因,phaC1Bc和phaC2Bc,在此缺失突变株中进行了表达.结果表明聚羟基脂肪酸酯的合成能力较相同条件下的野生型大肠杆菌有所提高.更多还原

【Abstract】 An E. coli fadB (fatty acid degradation) mutant strain KM32B (fadB∷Tet) is constructed using a method which requires no cloning or secondary PCR. Two primers consisting of 70 bases each are used. The first 45 bases are complementary to the 3′ and 5′ of E. coli any target gene. The other 25 bases target the 3′ and 5′ of a resistant marker such as tetracycline Tet. The PCR product, which has the resistant marker flanked by 45 bases upstream and downstream of the target gene, is electroporated into E. coli strain KM32. PCR analysis of genomic DNA from the recombinant cells indicates that the whole region of fadB gene is deleted and replaced by Tet. The functional expression of two PHA biosynthesis genes from Burkholderia caryophylli AS 1.2741, phaC1Bc and phaC2Bc, in this mutant strain shows increasing PHA production abilities, while no PHA is detectable in E. coli KM32 harboring phaC1Bc or phaC2Bc respectively.更多还原