Background

Motivation

Three Aims:

Aim 1:

Aim 2:

Aim 3:

Methods and Protocol

• assembled into a Bacillus-E. coli shuttle
• high-level, but tightly controlled GFP reporter expression in tunable, cumate linear concentration-dependent manner
• A major advantage of this promoter system over other, commonly used expression system is that cumate (p-isopropyl benzoate) is inexpensive, not metabolized as a carbon source and works efficiently at low concentrations.

Process:

E. coli and Bacillus strains were cultured in Luria–Bertani (LB) media at 37 °C with a 220-rpm in a shaking incubator

Different concentrations of cumate were added to cultures as indicated to induce gene expression. Control cultures were grown without cumate.

The pCT5-bac1.8 and pCT5-bac2.0 plasmids were trans- f o r m e d i n t o B . s u b t i l i s 1 6 8 b y e l e c t r o p o r a t i o n