Difference between revisions of "Part:BBa K2924015"

 
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The promoter aldA was published as an oleic acid (C18:1) sensitive promoter enabling for the measurement of free fatty acids present in microorganisms.
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===Usage and Biology===
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In <i>Escherichia coli</i> the growth on fatty acids requires many different proteins which are repressed by the transcriptional factor FadR.<sup>1</sup> The long chain acyl-CoA ester is an effector on the transcriptional factor FadR for the regulation of fatty acid metabolism.<sup>2</sup>
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[[File:FadR_mechanism.png|600px|thumb|left|<i><b>Fig.1:</b> 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.</i>]]
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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<sub>aldA</sub> was isolated from the <i>Escherichia coli</i>  wild type genome.
  
 
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===Usage and Biology===
 
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===Characterization===
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The sensitivity of the promoter to fatty acids was tested with the reporter gene eYFP (<a href=”https://parts.igem.org/Part:BBa_E0030”>BBa_E0030</a>) in the composite part <a href=”https://parts.igem.org/Part:BBa_K2924017”>BBa_K2924017</a>. 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.</html>
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[[File:PaldA_eYFP_ABC.png|500px|thumb|left|<i><b>Fig.1:</b> 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.</i>]]
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The <a href="https://www.protocols.io/view/fluorescence-measurement-8f9htr6">measurement</a> showed that the P<sub>aldA</sub> promoter produced a high amount of eYFP when oleic acid (C18:1) was added to the culture. In addition, P<sub>aldA</sub> 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).
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===References===
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1: Mee-Jung Han, Jeong Wook Lee, Sang Yup Lee, and Jong Shin Yoo. <i>“Proteome-Level Responses of Escherichia coli to Long-Chain Fatty Acids and Use of Fatty Acid Inducible Promoter in Protein Production” </i> Hindawi Publishing Corporation Journal of Biomedicine and Biotechnology Volume 2008, Article ID 735101,12 pages
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2: D. P. Clark and J. E. Cronan Jr., <i>“Two-carbon compounds and fatty acids as carbon sources,”</i> in Escherichia coli and Salmonella: Cellular and Molecular Biology, F. C. Neidhardt, R.Curtiss III, J. L. Ingraham, et al., Eds., pp. 343–357, ASM Press,Washington, DC, USA, 1996

Latest revision as of 16:36, 21 October 2019


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 PaldA 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

The sensitivity of the promoter to fatty acids was tested with the reporter gene eYFP (BBa_E0030) in the composite part BBa_K2924017. 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 PaldA promoter produced a high amount of eYFP when oleic acid (C18:1) was added to the culture. In addition, PaldA 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).

















References

1: Mee-Jung Han, Jeong Wook Lee, Sang Yup Lee, and Jong Shin Yoo. “Proteome-Level Responses of Escherichia coli to Long-Chain Fatty Acids and Use of Fatty Acid Inducible Promoter in Protein Production” Hindawi Publishing Corporation Journal of Biomedicine and Biotechnology Volume 2008, Article ID 735101,12 pages

2: D. P. Clark and J. E. Cronan Jr., “Two-carbon compounds and fatty acids as carbon sources,” in Escherichia coli and Salmonella: Cellular and Molecular Biology, F. C. Neidhardt, R.Curtiss III, J. L. Ingraham, et al., Eds., pp. 343–357, ASM Press,Washington, DC, USA, 1996