Regulatory
gadA

Part:BBa_K318512

Designed by: Sarah R. Sandock   Group: iGEM10_Wisconsin-Madison   (2010-07-22)

gadA (pH promoter and RBS)


Usage and Biology

gadAp: A pH Sensitive Promoter. One of the striking abilities of E. coli is its ability to pass through the acidic conditions of animal digestive tracts---GadA and the promoters and proteins around it may play a role in this. Results of northern blot analysis (Tucker et al., 2002; Waterman et al., 2003) indicate that a particular glutamate decarboxylase (GadA) is up-regulated, most likely as a part of a glutamate to gamma-aminobutyrate reaction which exports intercelluar protons which is required for E. coli stationary phase cell survival of highly acidic (pH 2) conditions (Castanie-Cornet et al., 1999). Based on a high ratio of low pH:neutral pH expression and a wealth of available information, we chose to use GadA's promoter, gadAp, as a native-E. coli sensor for acidic conditions. In order to maximize pH-mediated response of the gadA promoter and minimize induction caused by other known responses (such as the stationary phase), we plan to run error-prone PCR followed by screening of clones for maximal, dedicated acid response.

Low pH/Stationary Phase Induction

This part was tested in BBa_K318513

Download part information (.zip) here.

Below are two figures of sample data:

PH 7 gadAp with controls.jpg

Figure 4. Lines represent OD600 and are associated with the right axis. Dots indicate RFU/OD600 and associated with the left axis. In this trial, maximum induction occurred at an OD600 of approximately 6 (right axis, associated with line) and yielded about 10000 RFU/OD. Note that immediately before maximum OD600 was reached, RFU/OD increased about 5-fold. Controls included for this trial were a lacI promoter + RBS + mRFP1 + TT construct in the same plasmid (pSB1A2) and a WT control of MG1655 cells. These controls and results indicate that (1) mRFP1 expression by gadAp at high OD600 is a function of the gadA promoter, not of promoters at large (lacI promoter did not express) or of the cells themselves (MG1655 did not fluoresce).

PH 5.5 gadAp with controls.jpg

Figure 5.This trial was run parallel to the pH 7 trial in Figure 4. It indicates that cells grown in LB media at pH 5.5 do not exhibit a drastically different promotion behavior from their gadA promoters than those grown in neutral media.

Additional data points.jpg

Additional data points

Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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