Difference between revisions of "Part:BBa K2014000"

Latest revision as of 00:33, 22 October 2016

pBAD-E15'UTR->sfGFP

Usage and Biology

pBAD-E15'UTR->sfGFP construct is derived from pBAD (Arashort1, BBa_K1741000) Escherichia coli K-12 arabinose promoter, which we fused with E1_5’UTR (Fig. 1). E1_5’UTR contains an additional ribosome binding site from gene 10 of bacteriophage T7. The new promoter controls the expression of sfGFP with a His-tag at its N-end. The fluorescent protein sfGFP is a marker of gene expression and protein synthesis/accumulation. Protein expression from all compared arabinose responsive promoters was induced in rich media with 0.4% L-arabinose.

Fig. 1 Synthetic evolution of E. coli arabinose induced promoters in our lab. pBAD-E15'UTR (Arashort1-E1) is a promoter without AraC ORF, with E1_5’UTR containing the additional ribosome binding site from gene 10 of bacteriophage T7.

pBAD-E15'UTR (Arashort1-E1) is most likely the strongest of all available arabinose promoters. Its activity was compared to wild-type version (AraC-pBAD, BBa_K1481002 ) and to previously constructed arabinose promoters: Arashort1 (pBAD, BBa_K1741000), Ara1-UTR (pBAD-M5'UTR, BBa_K2014003) during 6h E. coli DH5α culture in LB medium supplemented with 0.4% L-arabinose (Fig. 2, Fig. 3). The pBAD-E15'UTR (Arashort1-E1) promoter-5’UTR fusion enables much higher protein expression in comparison to four other versions of arabinose promoter-UTR versions. The addition of E1_5'UTR increases promoter’s activity approximately 10-times in E. coli cells, grown in standard LB medium.

Fig. 2. Inducibility of pBAD-E15’UTR (Arashort1-E1) and AraC-pBAD (AraWT) promoters in E. coli grown in LB medium containing 0,4% L-arabinose. The efficiency of promoters was compared based on relative fluorescence intensity of produced sfGFP measured every 1 h after induction. OD600 shows that the growth rate of E. coli in both compared cultures is similar.

Fig. 3. Comparison between pBAD-E_15’UTR (Arashort1-E1) and our four previous arabinose promoter versions in E. coli grown in LB medium, induced with 0,4% L-arabinose. E. coli DH5α with appropriate constructs containing sfGFP were cultured for 6h after induction with 0,4% L-arabinose . The efficiency of promoters was compared based on relative fluorescence intensity of produced sfGFP.

Fig. 4. Selection of CPEC assembled clones of the construct pBAD-E15'UTR->sfGFP which produces His-tagged sfGFP on M9 minimal medium with chloramphenicol (75ug/ml) and 0.4% arabinose.

New, improved arabinose promoter pBAD-E15'UTR ensures approximately 10-fold increase in promoter’s activity. The addition of E1_5'UTR enables much higher protein expression in comparison to four other arabinose promoter-UTR versions.

Arabinose promoters - legend:
AraC-pBAD– formerly called AraWT [BBa_K1481002]
pBAD- formerly called Arashort1 [BBa_K1741000]
pBAD-M5'UTR– formerly called Ara1-UTR [BBa_K2014003]

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. 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
3. 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

Sequence and Features