Difference between revisions of "Part:BBa K2014001"

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prha1-E15'UTR->htsfGFP

Usage and Biology

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.

Fig. 1. Synthetic evolution of E. coli rhamnose responsive promoters in our lab. prha1-E15'UTR contains E1_5’UTR with the additional ribosome binding site from gene 10 of bacteriophage T7.

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).

Fig. 2. Comparison between prha1-E15’UTR (Rha1-E1) and rhaBAD (RhaWT) E. coli DH5α culture in LB medium with rhamnose (0.4% final conc.). The efficiency of promoters was compared based on relative fluorescence intensity of produced sfGFP. OD600 shows that the growth rate of E. coli in both compared cultures is similar.

Fig. 3. Fluorescence intensity comparison between prha1-E15’UTR (Rha1-E1) and its wild-type version rhaBAD (RhaWT). E. coli DH5α cells, transformed with constructs producing sfGFP, were cultured for 6h in LB medium supplemented with 0.4% rhamnose.

Fig. 4. Comparison between prha1-E15’UTR (Rha1-E1) and T7 promoter. E. coli DH5α cells were transformed with Rha1-E1 construct producing sfGFP, E. coli BL21-DE3 were transformed with T7-sfGFP construct. Both were cultured for 6h in LB medium supplemented with 0.4% rhamnose or lactose respectively.

Fig. 5. Fluorescence intensity comparison between prha1-E15’UTR (Rha1-E1) and T7 promoter. Flask with E. coli BL21-DE3 cells culture, transformed with T7-sfGFP, was grown in LB medium containing 0.4% lactose and E. coli DH5α transformed with Rha1-E1 (BBa_K2014001) was grown in LB medium containing 0.4% rhamnose. Both constructs were assembled in the same pSB1C3 vector, cultures were grown in the same medium containing chloramphenicol (75 μg/mL) and the plasmid copy number per cell should be identical or at least similar.

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