![](https://parts.igem.org/images/partbypart/icon_generator.png)
Part:BBa_K2014001
prha1-E15'UTR->htsfGFP
prha1-E1_5'UTR->htsfGFP is composed of three elements:
• prha1 - a modified rhamnose induced promoter (Bba_K1741005) originating from E.coli K-12 genome with a C→T substitution introduced to remove EcoRI site;
• E1_5’UTR containing an additional ribosome binding site from gene 10 of bacteriophage T7;
• an open reading frame for sfGFP.
The fluorescent protein sfGFP is a marker of gene expression and protein synthesis/accumulation. Protein expression from all compared rhamnose responsive promoters was induced in rich media with 0.4% rhamnose.
prha1-E1_5'UTR (Rha1-E1) is approximately 15-times stronger than its wild-type version rhaBAD and 3-times stronger than T7 promoter from pET system.
Our group compared prha1-E15'UTR (Rha1-E1) promoter with wild-type rhamnose promoter version rhaBAD (RhaWT) and the results show that Rha1-E1 ensures approximately 15-fold increase in protein production (Fig. 2, 3).
The new prha1-E1_5’UTR (Rha1-E1) promoter provides approx. 3-times higher protein expression than T7 promoter from pET system (Fig. 4, 5 ), whilst being much more tightly controlled.
References:
1. Olins PO, Rangwala SH.; A novel sequence element derived from bacteriophage T7 mRNA acts as an enhancer of translation of the lacZ gene in Escherichia coli. J Biol Chem. 1989 Oct 15;264(29):16973-6.
2. Davis J.H., Rubin A.J., Sauer R.T.; Design, construction and characterization of a set of insulated bacterial promoters. Nucleic Acids Research, 2011, Vol. 39, No. 3 1131–1141
3. Haldimann A., Daniels L.L, Wanner B. L.; Use of New Methods for Construction of Tightly Regulated Arabinose and Rhamnose Promoter Fusions in Studies of the Escherichia coli Phosphate Regulon. Journal of Bacteriology, Mar. 1998, p. 1277–1286
4. Holcroft C.C, Egan S.M. Roles of Cyclic AMP Receptor Protein and the Carboxyl-Terminal Domain of the a Subunit in Transcription Activation of the Escherichia coli rhaBAD Operon. Journal of Bacteriology, June 2000, p. 3529–3535
5. Giacalone M.J. et.al., Toxic protein expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter system. BioTechniques 40:355-364 (March 2006)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Unknown
None |