A Multi-Layer-Controlled Strategy for Cloning and Expression of Toxin Genes in Escherichia coli

Abstract

Molecular cloning and controlled expression remain challenging when the target gene encodes a protein that is toxic to the host. We developed a set of multi-layer control systems to enable cloning of genes encoding proteins known to be highly toxic in Escherichia coli and other bacteria. The different multi-layer control systems combine a promoter-operator system on a transcriptional level with a riboswitch for translational control. Additionally, replicational control is ensured by using a strain that reduces the plasmid copy number. The use of weaker promoters (such as P_BAD or PfdeA) in combination with the effective theophylline riboswitch is essential for cloning genes that encode notoriously toxic proteins that directly target translation and transcription. Controlled overexpression is possible, allowing the system to be used for evaluating in vivo effects of the toxin. Systems with a stronger promoter can be used for successful overexpression and purification of the desired protein but are limited to toxins that are more moderate and do not interfere with their own production.

Keywords: cloning; replicational control; riboswitch; toxins; transcriptional control; translational control.

Figure 1

Schematic overview of transcriptional and translational layers of control for the four systems developed in this study. Different promoters and riboswitches were combined to control the toxin’s (red) gene expression. (A) PfdeA-RS_B12 combination. The naringenin-inducible promoter PfdeA in light blue is positively regulated by the FdeR activator (grey) in presence of naringenin and is combined with the negatively regulated vitamin B₁₂ riboswitch (RS_B12, green). Only in absence of vitamin B₁₂, the ribosome binding site (RBS, orange) is accessible for ribosome binding. (B) Ptac-RS_B12 combination. The IPTG-inducible tac promoter (Ptac) in light purple is repressed by binding of the LacI repressor (brown) to the lac operator (lacO, purple) in absence of IPTG. Presence of IPTG allows transcription initiation. This is combined with the B₁₂ riboswitch. (C) Ptac-RS_theo combination. The lacI/lacO operator system is combined with the positively regulated synthetic theophylline riboswitch (RS_theo, yellow). Presence of theophylline (blue) alters the riboswitch conformation, allowing access to the RBS and therefore translation. (D) P_BAD-RS_theo combination. The arabinose-inducible promoter P_BAD (light green) is positively regulated through the binding of the AraC activator (magenta) to the I1 and I2 sites when bound to arabinose (turquoise). Combination with the RS_theo allows tight regulation of the toxic gene.

Figure 2

Spot test of serial diluted toxins on plates with or without inducing agents for the PfdeA—RS_B12 strategy. E. coli EPI400 harboring the successfully cloned toxins P1Doc, P1Doc^H66Y, EcMazF, EcMazF^E24A, EcParE2, VcHigB2, barnase and FCcdB on the pJYP1 vector was spotted on LB agar plates supplemented with ampicillin and additionally vitamin B₁₂ (A, OFF-state) or naringenin (B, ON-state) or a combination of both (C, partly induced) or none of the additional agents (D, partly induced).

Figure 3

Logarithmic normalized overview of cell growth observed for the PfdeA-RS_B12 strategy. Colonies are counted from the spot test on different LB ampicillin plates for E. coli EPI400 harboring toxins P1Doc, P1Doc^H66Y, EcMazF, EcMazF^E24A, EcParE2, VcHigB2, barnase and FCcdB. Raw colony count data were normalized to fraction survival for comparison of the OFF-state (vitamin B₁₂ and no naringenin, black bars) to the ON-state (naringenin and no vitamin B₁₂, grey bars).

Figure 4

Spot test of serial diluted toxins on plates with or without inducing agents for the Ptac—RS_theo strategy. E. coli EPI400 harboring the successfully cloned toxins P1Doc*, P1Doc^H66Y, EcMazF, EcMazF^E24A, EcParE2, VcHigB2, barnase* and FCcdB* on the pJYP3 vector were spotted on LB agar plates supplemented with ampicillin (A, OFF-state) and additionally theophylline and IPTG (B, ON-state) or IPTG alone (C, partly induced) or theophylline alone (D, partly induced).

Figure 5

Logarithmic normalized overview of cell growth observed for the Ptac—RS_theo strategy. Colonies are counted from the spot test on different LB ampicillin plates for E. coli EPI400 harboring toxins P1Doc, P1Doc^H66Y, EcMazF, EcMazF^E24A, EcParE2, VcHigB2, barnase and FCcdB. Raw colony count data were normalized to fraction survival for comparison of the OFF-state (no theophylline and no IPTG, black bars) to the ON-state (theophylline and IPTG, grey bars).

Figure 6

Spot test of serial diluted E. coli EPI400 harboring pJYP4_VcParE2 on plates with or without inducing agents for the P_BAD—RS_theo strategy. E. coli EPI400 harboring pJYP4_VcParE2 was spotted on (A, OFF-state) LB agar plates supplemented with ampicillin and (B, ON-state) theophylline and arabinose.

Figure 7

Small-scale expression of P1Doc and P1Doc^H66Y using the Ptac—RS_theo strategy. Samples of P1Doc* and P1Doc^H66Y before induction (BI) and four hours after induction (t = 4) were analyzed via 20% SDS-PAGE (left) and anti-histidine Western blot (right).

Figure 8

IMAC purification of P1Doc^H66Y (blue) and P1Doc* (red) using the Ptac—RS_theo strategy. Fractions collected during IMAC elution (A,B) were analyzed through 20% SDS-PAGE (C,D) and anti-histidine Western blot (E,F). The blue and red arrows respectively indicate P1Doc^H66Y and P1Doc* (14.6 kDa).