"
Team:Northwestern/detectpH
From 2013.igem.org
Detection of pH Levels
In order to negate pH drops after meals, we needed a way to detect the surrounding pH. We searched various methods of how different bacteria detect pH. However, we concluded that it would be better to search if E. Coli, our model bacteria, has any inherent mechanisms to detect pH levels. This way we don't need to insert genes that code for the all proteins involved in the pathway since it is already there. We just need to extract the promoter sequence and use it for our plasmid. In Gene Expression Profiling of the pH Response in Escherichia Coli (2002) , certain genes showed increased activity under low pH of 5.5, which is also the pH threshold for tooth enamel demineralization. Below is a table of changed gene expression and the red circles are the genes that had especially strong expression levels.
Figure 1. Statistically significant genes at pH 5.5 The significance was determined as induction ≥3 standard deviations from the mean (no change in expression) & P value of ≤ 0.05 as determined by t-test.
Out of the three genes (Asr, gadA, and hdeA), Asr and GadA were more isolated compared to hdeA. Our project looked into these two genes for pH detection. Both have different mechanisms of sensing pH. Asr works through the PhoBR operon system.
Figure 2. PhoB, when phosphorylated by PhoR, would form homodimers and bind to the consensus pho-box sequence involved in the corresponding promoter and activate downstream genes expression.
For GadA, GadA forms an operon with GadX. GadE activates GadA and GadB. Binding of RcsB and gadE as a heterodimer to the GAD box activates gadA transcription.
Figure 3. GadA Mechanism
