The gene of interest is cloned into the pBAD vector and maintained in growth medium lacking L-arabinose. This reduces plasmid instability that could result from the expression of proteins of interest that may be harmful to host cells. Afterwards, expression of the gene of interest can be induced by the addition of L-arabinose to the medium.
Our pBAD vectors contain the bidirectional araBAD promoter, which drives expression of both the gene of interest and the regulatory protein AraC. In the absence of L-arabinose, AraC dimerizes to form a loop in the promoter region, blocking transcription. When L-arabinose is added, AraC changes conformation and binds to alternate sites in the promoter, activating transcription.
Addition of glucose to the growth medium can further suppress basal expression due to a reduction in cellular cAMP levels. In glucose-free medium, cAMP levels are high, and a cAMP-CRP (catabolite activator protein) complex binds to the pBAD promoter. This association is required for promoter activity, so addition of glucose will robustly repress expression of the gene of interest. This is particularly useful when the gene of interest is toxic or inhibits bacterial growth.
All custom pBAD vectors will be supplied in an E. coli strain designed to maximize plasmid integrity (such as Stbl3). To express the protein of interest, the plasmid can be transferred to a host strain lacking genes for L-arabinose catabolism (such as TOP10). This allows efficient and stable gene activation without reduction in the L-arabinose concentration over time. For genes of interest that require tight suppression of basal expression by glucose, the LMG194 host strain should be used. This strain is capable of growth on minimal defined medium (RM medium), allowing additional repression of the araBAD promoter by glucose.
