Screening of differentially regulated Escherichia coli promoters in dual-species co-cultures.
Tee, Carolyn Sze Yeang.
Date of Issue2009
School of Biological Sciences
Commercially-obtained Escherichia coli (E. coli) promoter library fused upstream of the green fluorescent protein (GFP) gene (commercial GFP library), previously constructed promoter-prototype combined library with known promoters collection, and promoter capture library, in which the upstream of AsRed2 gene was fused with digested genomic DNA fragments, were used to monitor the promoter activities of E. coli in dual species co-cultures with the cellular ratio 1:1 in different conditions. After checking the performances of the libraries, the promoter-capture libraries were found to be below detection limits. While the reporter levels of the promoter-prototype libraries were higher than that of the promoter capture libraries. The commercial GFP library which comprised of ~1800 promoters demonstrated reporting level above detection limit. Therefore, the commercial GFP library was chosen to co-culture with Klebsiella pneumoniae and Enterococcus faecalis in minimal or rich media-representing different osmolarities, and at different temperature- 30°C and 37°C. 6 differentially regulated promoters were identified, sequenced and analyzed, such as lacI gene and wrbA gene, whose products are a DNA-binding transcriptional repressor and a Trp repressor binding protein, respectively. This illustrates that E. coli with different genetic background demonstrate differences in gene expression upon interaction with non-E. coli species under different culture conditions.
Final Year Project (FYP)
Nanyang Technological University