Regulatory
p(osmY)
Part:BBa_J45992:Design
Designed by Stephen Payne Group: iGEM2006_MIT (2006-07-11)
From partsregistry.org
Full-length stationary phase osmY promoter
Assembly Compatibility:
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 1
- 21INCOMPATIBLE WITH RFC[21]Illegal prefix found in sequence at 1
- 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 1
- 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 1
Design Notes
- We considered different designs for stationary phase regulation of odor production. All designs focused on using transcriptional control devices to regulate the odor enzyme generators and thus odor production. One candidate design for stationary phase regulation was to use a transcriptional promoter that is only active in stationary phase. Several E. coli promoters that are primarily active in stationary phase have been previously characterized [1, 2]. Thus, we could evaluate various stationary phase promoters as potential growth phase dependent transcriptional control devices.
- We evaluated three stationary phase promoters as potential stationary phase dependent transcriptional control devices. We first tested the promoter that controls transcription of rpoS in E. coli [3]. The rpoS gene encodes σS factor, a transcription factor known to be present at higher levels during late exponential phase and early stationary phase [4, 5]. Our preliminary experiments with a plasmid encoding the rpoS promoter controlling transcription of green fluorescent protein (GFP, plasmid pBS-rrnBTrpoSpUV) demonstrated that the rpoS promoter did not produce significantly higher fluorescence in stationary phase than during exponential growth [6]. We then identified a second E. coli stationary phase promoter for evaluation: the promoter that controls transcription of osmY [7, 8]. Expression of osmY is dependent on σS in vivo [3]. Schellhorn et al. previously demonstrated that an \textit{osmY::lacZ} fusion generated the highest transcriptional signal in stationary phase as compared to nine other σS-dependent promoter-lacZ fusions [1, 2]. In addition, the osmY::lacZ fusion generated only a small transcriptional signal during exponential growth.
- Constructed by PCR
Template
Escherichia coli strain MG1655
Forward Primer
5'- GTT TCT TCG AAT TCG CGG CCG CTT CTA GCT GGC ACA GGA ACG TTA TC - 3'
Reverse Primer
5'-GTT TCT TCC TGC AGC GGC CGC TAC TAG TAT TGT TAA ATA TAG ATC ACA ATT TTG AAA CCG- 3'
Source
- Promoter controlling expression of osmY from Escherichia coli (GenBank S67278)
References
- Schellhorn HE, Audia JP, Wei LI, and Chang L. Identification of conserved, RpoS-dependent stationary-phase genes of Escherichia coli. J Bacteriol 1998 Dec; 180(23) 6283-91. pmid:9829938.
- Hengge-Aronis R, Lange R, Henneberg N, and Fischer D. Osmotic regulation of rpoS-dependent genes in Escherichia coli. J Bacteriol 1993 Jan; 175(1) 259-65. pmid:8416901.
- Lange R and Hengge-Aronis R. Identification of a central regulator of stationary-phase gene expression in Escherichia coli. Mol Microbiol 1991 Jan; 5(1) 49-59. pmid:1849609.
- Tanaka K, Takayanagi Y, Fujita N, Ishihama A, and Takahashi H. Heterogeneity of the principal sigma factor in Escherichia coli: the rpoS gene product, sigma 38, is a second principal sigma factor of RNA polymerase in stationary-phase Escherichia coli. Proc Natl Acad Sci U S A 1993 Apr 15; 90(8) 3511-5. pmid:8475100.
- Funabashi H, Haruyama T, Mie M, Yanagida Y, Kobatake E, and Aizawa M. Non-destructive monitoring of rpoS promoter activity as stress marker for evaluating cellular physiological status. J Biotechnol 2002 Apr 25; 95(1) 85-93. pmid:11879714.
- Yim HH and Villarejo M. osmY, a new hyperosmotically inducible gene, encodes a periplasmic protein in Escherichia coli. J Bacteriol 1992 Jun; 174(11) 3637-44. pmid:1317380.
- Yim HH, Brems RL, and Villarejo M. Molecular characterization of the promoter of osmY, an rpoS-dependent gene. J Bacteriol 1994 Jan; 176(1) 100-7. pmid:8282684.
- Wise A, Brems R, Ramakrishnan V, and Villarejo M. Sequences in the -35 region of Escherichia coli rpoS-dependent genes promote transcription by E sigma S. J Bacteriol 1996 May; 178(10) 2785-93. pmid:8631665.
- Makinoshima H, Nishimura A, and Ishihama A. Fractionation of Escherichia coli cell populations at different stages during growth transition to stationary phase. Mol Microbiol 2002 Jan; 43(2) 269-79. pmid:11985708.
- Vijayakumar SR, Kirchhof MG, Patten CL, and Schellhorn HE. RpoS-regulated genes of Escherichia coli identified by random lacZ fusion mutagenesis. J Bacteriol 2004 Dec; 186(24) 8499-507. doi:10.1128/JB.186.24.8499-8507.2004 pmid:15576800.
Neidhart, Frederick C. Escherichia coli and Salmonella. Washington: ASM Press, 1996.
