Difference between revisions of "Part:BBa K1668004"

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ermEp (a strong constitutive promoter in S. avermitilis)

ermEp is a strong constitutive promoter in various S. avermitilis strains. It should be noticed that ermEp can only be expressed in S.avermitilis strains instead of Escherichia coli or any other chassis.

Characterization

Background

Overview

The ermE promoter was originally characterised by cloning the entire putative promoter region upstream of the ermE gene of Saccharopolyspora erythraea in front of a kanamycin resistance gene (neo) in a replicative vector in Streptomyces lividans TK24.

Origin

The ermE promoter region contains two different promoters, ermEp1 and ermEp2. It was reported that a TGG deletion in the 35 region of the ermEp1 promoter resulted in a stronger variant called ermE* (ermEp2 and ermEp1 ΔTGG). The promoter strength was indirectly assessed according to the enzymatic activity of the reporter protein GUS. The ermE and ermE* promoters were approximately 1.8 times stronger than the ermEp1 and ermEp1* promoters. However, no significant difference was detected between the strengths of the native ermE promoter and its variant ermE* or between the ermEp1 and the ermEp1* promoter.

RESULTS

Gel electrophoretic analysis

Fig.2 Gel electrophoretic analyses of PCR products (A) and selected examples of cloned products of seamless assembly reaction (B)

In Fig.2, (A) 5-μl samples of the PCR products for ermEp, (B) 5-μl samples of the bacteria solution PCR products were loaded onto a 1% BioRad Ready Agarose Mini Gel, then subjected to AGE. See (protocol) for AGE parameters. The DNA size standards was the DL1,000 DNA Marker (M; Takara, Cat#3428A). Bands were visualized with a Shanghai Peiqing JS-380A Fluorescence Imager. PCR products, positive clones and negative clones are indicated.

DNA sequencing

We have sequenced the parts with standard primers VF2 and VR. The sequence of the 155bp part shows 100% agreement with the desired sequence.

REFERENCE

Siegl, T., et al. (2013). "Design, construction and characterisation of a synthetic promoter library for fine-tuned gene expression in actinomycetes." Metab Eng 19: 98-106.
Bibb, M. J., et al. (1994). "The mRNA for the 23S rRNA methylase encoded by the ermE gene of Saccharopolyspora erythraea is translated in the absence of a conventional ribosome-binding site." Mol Microbiol 14(3): 533-545.