Difference between revisions of "Part:BBa K1231001"

Revision as of 03:09, 17 October 2019

GadA-RBS is a pH sensitive promoter

GadA-RBS is a pH sensitive promoter that has been shown to initiate transcription at low pH.

Usage and Biology

The gadA gene is a part of the gad system, which is an acid-inducible glutamate decarboxylase-based acid resistance system that enables the survival of E. coli under acid stress conditions. GadA is a gene encoding a glutamate decarboxylase. The process of decarboxylating glutamate consumes protons that leach into the cell under acid stress. In this manner, the gad system manages protons that would otherwise drop the cellular pH below levels at which E. coli could survive.

The function of the gad system explains why these genes experience high transcription levels at low pH. Despite this functional explanation, modes of transcriptional control over the gad system are rather complex. Transcriptional factors RpoS, cyclic AMP receptor protein, HN-S and EvgA all play a role in transcriptional regulation. Transcriptional regulators GadW and GadX also affect induction of the gad system through intricate interactions both with the GadA promoter region, as well as with one another. Due to this complexity, our strategy was to characterize the gadA promoter empirically.

We found that a previous iGEM team (University of Wisconsin-Madison 2010) had used the GadA promoter. We contacted the professor who advised said iGEM team and requested their GadA promoter part since it was not in stock at iGEM HQ. However, they were unable to provide us with such a part, so we obtained the promoter via genomic PCR on E. Coli. Through our pH fluorescence testing, we were able to gather results on GadA promoter's reactivity to more pH levels than what the University of Wisconsin-Madison team was able to accomplish. We were therefore able to better understand the pH promoter and how it functions.

In 2019, our team (HUST-China) have characterized this part again and the results showed that the GadA promoter has higher expression in pH=7 and pH=6 than pH=5, and we put our data below. In experiment, we use plate reader and the iGEM 2019 Plate Reader Abs600 (OD) Calibration and iGEM 2019 Plate Reader Fluorescence Calibration. But our data is quite different from the data Northwestern showed. So we decided to do this experiment again, but we have no result yet.

Sequence and Features