Homogeneous expression of the PBAD promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter

Abstract

Genes placed under the control of the arabinose-inducible araBAD promoter (P BAD ) of Escherichia coli are expressed in an all-or-none fashion, in which the percentage of induced cells in the population, rather than the degree of induction in individual cells, varies with the concentration of arabinose in the culture medium. Previous work showed that all-or-none gene expression from P BAD was due to the arabinose-dependent expression of the gene encoding the low-affinity high-capacity transporter (araE), and that expression of heterologous genes from P BAD in individual cells could be regulated by placing the araE gene under control of an arabinose-independent promoter. Based on these results, two expression systems were developed to allow regulatable control of genes under control of P BAD . In one system, the native araE promoter on the chromosome was replaced by constitutive promoters of different strengths. In the second system, the araE gene under control of the same constitutive promoters was placed on a medium-copy plasmid. Both systems allow regulatable expression of a plasmid-borne P BAD -controlled heterologous gene and a homogeneous population of cells over a wide range of arabinose concentrations. While the degree of induction varied slightly with the strength of the constitutive promoter, expression was affected most by the arabinose concentration.

The Regulatory Roles of the Galactose Permease and Kinase in the Induction Response of the GAL Network in Saccharomyces cerevisiae

“…Specifically, the positive feedback control loop regulating the expression of the galactose permease is a necessary component of the observed all-or-none response of this network. This has been demonstrated in simpler bacterial networks such as the arabinose and lactose operons (17). The results demonstrate a significant negative population irrespective of Gal2p regulation except at high galactose concentrations.…”

“…For example, several groups have altered the network connectivity of the arabinose metabolic network to exhibit a more tunable, homogenous response from arabinose-inducible promoter systems in Escherichia coli as opposed to its native, all-ornone response (17,18). This tunable promoter system was designed by altering the regulation of the arabinose transporter gene from autocatalytic control to constitutive or researcher-controlled systems that resulted in a more linear induction response (17). In another recent study, the feedback loops of Gal3p and Gal80p were implicated in the memory response of the GAL network to growth history in S. cerevisiae (14).…”

Improvement of DNA minicircle production by optimization of the secondary structure of the 5′-UTR of ParA resolvase

“…The construction of the strains BWAA, BW1P, and BW2P, with enhanced uptake for the arabinose inducer (Khlebnikov et al 2001) and deficiency in endA and recA genes that increases the amount and the quality of pDNA (Phue et al 2008;Gonçalves et al 2013), was essential for a higher minicircle production. When ParA was expressed from low-copy number plasmids in the BWAA strain, full recombination of the model parental plasmid pMINI was obtained within 2 h, but no effect of the 5′-UTR was detected (Fig.…”

“…The E. coli strain BW27783 [F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ − , Δ(araH-araF)570(::FRT), ΔaraEp-532::FRT, φP cp8 araE535, rph-1, Δ(rhaDrhaB)568, hsdR514], originally designed for improved arabinose uptake (Khlebnikov et al 2001) was purchased from the Coli Genetic Stock Center (CGSC) at Yale (USA) and used to generate strains BWAA, BW1P, and BW2P as described next.…”

“…We reasoned that by changing the nucleotide sequence of this 5′ untranslated region (5′-UTR), potential hairpins that might be causing ribosomes to stall could be destabilized, thus increasing translation of mRNA and ParA levels and ultimately improving recombination of parental plasmids into minicircles. In our model system, the ParA resolvase was placed under the control of the arabinose-inducible P BAD /araC promoter (Smith and Schleif 1978) and strains derived from BW27783, which was designed for improved uptake of arabinose (Khlebnikov et al 2001), were used for parental plasmid production and recombination. Figure 1a shows the nucleotide sequence of the original araBAD regulatory region.…”