Difference between revisions of "Part:BBa K1231001"

Latest revision as of 12:15, 20 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.

Characterization from HUST-China 2019

In 2019, our team (HUST-China) have characterized this part again and the results showed that the GadA promoter has approximately same expression in pH=7, pH=6 and pH=5, and we put our data below. The part we characterized in experiment was synthesised by company, using the gene sequence from this page.We linked the part fragment with gfp gene by overlap extension PCR and transferred it into E. coli to express gfp gene，and cultured it in pH gradient medium.We designed five kinds of medium, which pH=3\4\5\6\7. Because there was no bacterial growth in pH=3 and 4, we abandoned the data for these two. As for measurement, we used plate reader and obeied the iGEM 2019 Plate Reader Abs600 (OD) Calibration and iGEM 2019 Plate Reader Fluorescence Calibration. Our data showed that the real result is not fit for our expectation.

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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 <partinfo>BBa_K1231001 short</partinfo>
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 GadA-RBS is a pH sensitive promoter that has been shown to initiate transcription at low pH.
-<!-- Add more about the biology of this part here
+https://static.igem.org/mediawiki/2013/8/8d/NorthwesternGRG_Fl_nom.png
+<!-- Add more about the biology of this part here -->
 ===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.
+===Characterization from HUST-China 2019===
+In 2019, our team (HUST-China) have characterized this part again and the results showed that the GadA promoter has approximately  same expression in pH=7, pH=6 and pH=5, and we put our data below. The part we characterized in experiment was synthesised by company, using the gene sequence from this page.We linked the part fragment with gfp gene by overlap extension PCR and transferred it into E. coli to express gfp gene，and cultured it in pH gradient medium.We designed five kinds of medium, which pH=3\4\5\6\7. Because there was no bacterial growth in pH=3 and 4, we abandoned the data for these two. As for measurement, we used plate reader and obeied the iGEM 2019 Plate Reader Abs600 (OD) Calibration and iGEM 2019 Plate Reader Fluorescence Calibration. Our data showed that the real result is not fit for our expectation.
+https://2019.igem.org/wiki/images/c/ce/T--HUST-China--pH_responsing_system.png
 <!-- -->