Part:BBa_K2924015

Promoter aldA from the Escherichia coli genome

The promoter aldA was published as an oleic acid (C18:1) sensitive promoter enabling for the measurement of free fatty acids present in microorganisms.

Usage and Biology

In Escherichia coli the growth on fatty acids requires many different proteins which are repressed by the transcriptional factor FadR.¹ The long chain acyl-CoA ester is an effector on the transcriptional factor FadR for the regulation of fatty acid metabolism.²

Fig.1: Regulation of the fatty acid metabolism by the transcriptional factor FadR. FadR recognizes its cognate binding site (white), thereby repressing transcription. Upon binding of an Acyl-CoA to FadR, the promoter region is freed, enabling gene expression.

After adding oleic acid (C18:1) many proteins showed an altered expression level and new proteins like AldA were synthesised. The promoter for aldA was also used for the production of green fluorescent protein (GFP).1 P_aldA was isolated from the Escherichia coli wild type genome.

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]

Characterization

Fig.1: Regulation of the fatty acid metabolism by the transcriptional factor FadR. FadR recognizes its cognate binding site (white), thereby repressing transcription. Upon binding of an Acyl-CoA to FadR, the promoter region is freed, enabling gene expression.

After adding oleic acid (C18:1) many genes showed an altered expression level and new proteins like AldA were synthesised. The promoter for aldA was also used for the production of green fluorescent protein (GFP) as a reporter ¹. P_aldA was isolated from the Escherichia coli wild type genome.

The sensitivity of the promoter to fatty acids was tested with the reporter gene eYFP (Part:BBa_E0030) ³. Different long-chain fatty acids, like palmitic acid, stearic acid and oleic acid were used for the characterization. Also different concentrations from 0.01 mM to 1 mM were used of the fatty acid palmitic acid and stearic acid. For the fatty acid oleic acid, the concentrations from 0.5 mM to 10 mM were used.

Fig.1: Response of paldA+eYFP (yellow) to different chain lengths of fatty acids compared to an empty vector control (gray). Plot A presents the response for palmitic acid, plot B for stearic acid and plot C for oleic acid. The fluorescence was measured at an excitation wavelength from 497 nm and an emission wavelength from 540 nm.

The measurement showed that the P_aldA promoter produced a high amount of eYFP when oleic acid (C18:1) was added to the culture. In addition, P_aldA can also detect stearic acid (C18:0) the saturated form of oleic acid, as well as palmitic acid(C16:0), although with a weaker absolute fluorescence value than oleic acid (C18:1).