A single vector system for tunable and homogeneous dual gene expression in Escherichia coli

Abstract

Expression of recombinant genes can be controlled using inducible promoters. However, the most commonly used IPTG- and arabinose-inducible promoters result in an 'all-or-nothing' response, leading to fully induced and uninduced bacterial subpopulations. Here, we investigate whether appropriate modifications to these promoter systems can be combined into a single vector system, enabling homogenous expression of two genes of interest that can be precisely tuned using inducer concentration. We show that modifications of positive feedback loops related to inducer uptake result in homogeneous gene expression in both the T7 lactose and pBAD arabinose systems. Furthermore, these two modified systems were combined into a single vector, pRAT-sfGFP that provides the desired tunable expression of two genes of interest. Finally, we test this single-vector system as a tool for studying two-component genetic circuits, using toxin-antitoxin modules as model systems. This novel low-copy single vector expression system opens up new possibilities for investigating the function of two-component bacterial genetic circuits.

Keywords: Arabinose; Dual tunable gene expression; HigBA2; IPTG; Phd/Doc; Toxin-antitoxin; Two-component genetic circuits.

Fig. 1

Population-homogeneous gene expression using IPTG and arabinose. (A) Top panel shows the percentage of IPTG-induced sfGFP expression measured using flow cytometry. Middle panel shows florescence intensity as a function of inducer concentration for expression of sfGFP in Tuner[DE3] cells measured in plates (black symbols) or by flow cytometry (white symbols, averages of three colonies, error bars indicate standard error of measurement). Bottom panel shows flow cytometry population distribution curves as a function of IPTG concentration for colony 1. (B) Percentage of arabinose-induced cells (top panel) and sfGFP fluorescence intensity (middle panel) as a function of arabinose concentration. Cells were either measured directly in plates (black symbols) or using flow cytometry (white symbols). Bottom panel shows the corresponding population distribution curves as a function of arabinose concentration for colony 1.

Fig. 2

IPTG and arabinose show no significant cross-talk. (A) sfGFP fluorescence intensity induced by IPTG (left) or arabinose (right) in presence of the second interfering inducer. (B) Population distribution curves for sfGFP induction by 0.1 and 1 mM arabinose (black) in presence of second inducer IPTG (gray, light gray). (C) Population distribution curves for sfGFP induction by 0.01, 0.05, 0.1 and 10 mM IPTG (black) in presence of second inducer arabinose (gray, light gray).

Fig. 3

Different ratios of gene expression can be achieved by varying inducer concentration or induction times. (A, B) Normalized fluorescence ratios dor the sfGFP-A/sfGFP-I expression measured separately at 3 h post-induction with both inducers. (C) Normalized fluorescence ratios for the sfGFP-A/sfGFP-I expression measured separately for cells grown overnight in presence of IPTG, while arabinose was induced for 3 h.

Fig. 4

Design of the pRAT-sfGFP scaffold vector and its application for studying two-component genetic circuits. (A) pRAT-sfGFP vector map shows sites of kanamycin resistance gene (KanR, dark gray), p15A ori (white), repressors lacI and AraC (gray), reporter protein sfGFP (green), arabinose promoter (PBAD), lactose operator (lacO), T7 promoter (PT7) and 4 pairs of homology regions (HR1-8) for Golden Gate cloning using BsaII. (B) Normalized fluorescence measurements of sfGFP upon induction of antitoxin (HigA2, Phd, left) or toxin (HigB2, Doc, right). Values in absence of inducer are indicated as a dotted line (no inducer). Toxin induction was performed in the presence of antitoxin, at the IPTG concentration level resulting in fluorescence values indicated in green (dot/line). (C) Immunodetection using ELISA of HigA2 (left) and HigB2 (right) following induction using IPTG and arabinose, respectively. Black symbols represent absorbance measurements at 450 nm (A450) whereas white and gray symbols represent calculated protein concentration.