In the laboratory, IPTG serves as one of the most effective molecular inducers of transcriptional activity and therefore genetic elements responsive to this inducer are common. Commercialized pET vectors used a bacteriophage T7 late promoter upstream of the gene or genes of interest. This promoter is only recognized by the T7 polymerase which is under control of the IPTG-inducible lacUV5 promoter. This system creates large amounts of mRNA and, often the desired protein in high concentrations (40 to 50% of the total cell protein). However, has drawbacks such as high level of mRNA can cause cell death or possibly results in plasmid or expression instability. The pET plasmids also are toxic to E. coli in the presence of IPTG(Miroux and Walker, 1996).
Additionally, IPTG is moderately expensive; this makes many approaches that utilizes this inducer financially impractical at an industrial scale with specific biotechnological products(Briand et al., 2016). To make the production of PHA plastics competitive with petroleum-based plastics, as many costs as possible will have to be reduced. Therefore, developing an alternate strategy for the induction of transcription of the genes that code for the enzymes in the pathway of production of the product represents a hurdle to be overcome.
LUDOX CL-X (45% colloidal silica suspension) and water replicates were pipetted into a plate to get an absorbance reading. These readings were used to create a ratio to convert absorbance to OD.
Figure 1 The glucose sensing toggle switch architecture showing individual parts. Toggle switch contains the LacI responsive promoter (trcp), which expresses TetR and mCherry, and the TetR responsive promoter (tetOp) which expresses lacI and gfp. The two transcription are under the control of the opposite’s promoter. The (cAMP) receptor promoter (crpp) activates transcription of lacI when glucose levels become low, turning the switch (ON). Reporter proteins (GFP and RFP) show whether the toggle switch is on or off. Image modified from Bothfeld et al.(Bothfeld et al., 2017)
