Promoter Characterization of the Creatininase Gene from Pseudomonas putida RS65 in E. coli

• Main Authors: Lu Yun, 雲露
• Other Authors: Wen-Hsiung Liu
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/82015687078104165160

碩士 === 國立臺灣大學 === 微生物與生化學研究所 === 92 === Creatininase is used in quantitative analysis of creatinine in urine and serum on medical test, to evaluate the function of kidney. A 1.1 kb gene fragment encoding creatininase cloned by pUC18 from Pseudomonas putida RS65 can be constitutively expressed in E. coli. After deleting the lac promoter on the recombinant plasmid consistent of pUC18 and 1.1 kb creatininase gene fragment, the creatininase activity can still be detected in the E. coli transformants, showing that the 5’-flanking region of this creatininase gene has promoter function. Analyzing the 5’-flanking region of the creatininase gene by using enhanced green fluorescent protein (EGFP) as reporter gene, we found that there is no EGFP activity detected in E. coli transformants. By using its own creatininase coding sequence as reporter gene and progressively deleting the 160 bp 5’-flanking region fragment to analyze its promoter function, we found that there exist probable cis-elements at the range of from the position -100 to -80 bp and from –80 to -40 bp. But point mutation on these areas didn’t result in any obvious creatininase expression differences. On the other hand, by partially deleting this 160 bp 5’-flanking region fragment, creatininase activity of deletion of the range from position –102 to -41 bp is higher than that of the original 160 bp fragment about 26%; and about 7% higher after deleting the range from -120 to -30 bp. According to the results of searching on the online database, we found that the direct repeat at the position -128 to –111 bp and -37 to -20 bp might be a protein binding motif in the creatininase promoter region and in charge of its main function. And other sequence between position –110 and –36 bp in this 160 bp 5’-flanking region fragment from P. putida probably would be recognized by positive or negative E. coli trans-elements at transcriptional level.