Overview

This year, XMU-China still devotes to making improvement for previous brick according to our project. Our improved part is L-rhamnose-inducible promoter (BBa_K914003).

Background

pRha (BBa_K914003) is a strictly regulated promoter, which was firstly registered in 2013 and has been widely used in stringently expression of GOI (gene of interest) (1-3). RhaS is a transcriptional activator of pRha, which will sense L-rhamnose and allosterically bind pRha to activate the transcription.

Design

In order to improve the induced expression level of L-rhamnose-inducible promoter BBa_K914003, an extra copy of rhaS gene was constitutively expressed and placed upstream the pRha, in which the eyfp gene (BBa_E0030) was chosen as the reporter to finally construct the two characterization circuits of original and improved parts (Fig. 1).

Fig. 1 Gene circuit illustration for improved pRha promoter system.

Result

Different sub parts of the two circuits were assembled into pSB4K5 plasmid backbone using Gibson assembly to get the composite part BBa_K4195109 and BBa_K4195191. The Gibson assembly reaction mixture was transformed into E. coli DH5α and E. coli BL21(DE3), then the correct colonies were confirmed by colony PCR (Fig. 2) and sequencing.

Fig. 2 The result of colony PCR.

Then, these colonies harboring the corrected plasmid were cultivated and induced to measure the expression level of EYFP (eyfp). Both the composite parts were induced under L-rhamnose (final concentration was 0.1 mg/mL) and the sterile water of same volume was added as the control. The fluorescence intensity (λ_Ex = 507 nm, λ_Em = 532 nm) and OD₆₀₀ was measured in 2 h after induction. The relative fluorescent unit (RFU) normalized to OD₆₀₀, RFU_EYFP/OD₆₀₀, was calculated to represent the expression level of EYFP in an average cell. We can obviously find that the gene circuit with rhaS showed higher level of fluorescence signal, even though a little bit of leakage expression was observed (Fig. 3).

Fig. 3 The comparison of normalized fluorescence intensity between BBa_K4195109 and BBa_K4195191.

What’s more, we also set a series of concentration gradients to compare different inducers’ effects on the improved L-rhamnose-inducible promoter. Learn more specific and detailed information in the parts page of this composite part (BBa_K4195109).

Reference

1. S. K. Kim, D. H. Lee, O. C. Kim, J. F. Kim, S. H. Yoon, Tunable Control of an Escherichia coli Expression System for the Overproduction of Membrane Proteins by Titrated Expression of a Mutant lac Repressor. ACS Synth. Biol. 6, 1766-1773 (2017).
2. A. Haldimann, L. L. Daniels, B. L. Wanner, Use of new methods for construction of tightly regulated arabinose and rhamnose promoter fusions in studies of the Escherichia coli phosphate regulon. J. Bacteriol. 180, 1277-1286 (1998).
3. Q. Li et al., A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli. Acta Biochim. Biophys. Sin. 53, 620-627 (2021).
4. C. L. Kelly et al., Synthetic Chemical Inducers and Genetic Decoupling Enable Orthogonal Control of the rhaBAD Promoter. ACS Synth. Biol. 5, 1136-1145 (2016).