Difference between revisions of "Part:BBa K4788002"
 
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This is a promoter module that can respond to oleic acid induction. And it has different response intensities for different oleic acid concentrations.
 
This is a promoter module that can respond to oleic acid induction. And it has different response intensities for different oleic acid concentrations.
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===Usage and Biology===
 
===Usage and Biology===
  
 
The FadR protein expressed by the FadR gene can bind to the operator FadO following the FadBA promoter, which inhibits the initiation of FadBA. However, oleoyl CoA metabolized from oleic acid can relieve this inhibition and promote the expression of FadBA promoter. This allows it to be induced by oleic acid for expression. In practical applications, we can change the number and position of FadO operators to change their response threshold to oleic acid to achieve more precise regulatory effects.
 
The FadR protein expressed by the FadR gene can bind to the operator FadO following the FadBA promoter, which inhibits the initiation of FadBA. However, oleoyl CoA metabolized from oleic acid can relieve this inhibition and promote the expression of FadBA promoter. This allows it to be induced by oleic acid for expression. In practical applications, we can change the number and position of FadO operators to change their response threshold to oleic acid to achieve more precise regulatory effects.
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===Characterization===
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In order to characterize the oleic acid inducer, we added mRFP after the oleic acid inducer for characterization, and we validated the response of the oleic acid inducer to gradient oleic acid by inducing the initiation of the oleic acid promoter under both aerobic and anaerobic conditions, as well as the operability of the part under anaerobic conditions. For detailed information, please refer to <html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_K4788008"> BBa_K4788008</a ></html>
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==Characterization results==
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To verify the effect of oleic acid inducer, we added mRFP gene after the oleic acid inducer gene to construct a reporter gene. We cultured engineering bacteria under two different environmental conditions, anaerobic and aerobic, and under different oleic acid induction conditions, and detected the expression effect of oleic acid inducers through normalized fluorescence signal intensity. As shown in Figure 1, the oleic acid inducer has a good response to gradient oleic acid, especially in the absence of oxygen, showing significant differences from the previous gradient at a 10% oleic acid gradient.
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<html>
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<figure style="text-align:center;">
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                <img style="max-width:700px;" src="https://static.igem.wiki/teams/4788/wiki/proof/fad-separated.jpg" alt="control">
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                <figcaption><b>Figure1:</b> Gradient oleic acid induced expression intensity of oleic acid starter under both anaerobic and aerobic conditions.</figcaption>
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              </figure>
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</html>
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Under the comparison of anaerobic and aerobic conditions, as shown in Figure 2, there was a slight difference in the expression level of the oleic acid inducer gene, but it did not show significant difference.
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<html>
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<figure style="text-align:center;">
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                <img style="max-width:700px;" src="https://static.igem.wiki/teams/4788/wiki/proof/fad-all.png" alt="control">
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                <figcaption><b>Figure 2:</b> Comparison of the expression intensity of gradient oleic acid induced oleic acid starter under anaerobic and aerobic conditions.</figcaption>
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              </figure>
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</html>
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===Reference===
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Ma Y, Zheng X, Lin Y, Zhang L, Yuan Y, Wang H, Winterburn J, Wu F, Wu Q, Ye JW, Chen GQ. Engineering an oleic acid-induced system for Halomonas, E. coli and Pseudomonas. Metab Eng. 2022 Jul;72:325-336. doi: 10.1016/j.ymben.2022.04.003. Epub 2022 May 2. PMID: 35513297.
  
 
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Latest revision as of 01:35, 12 October 2023


Oleic acid inducer

This is a promoter module that can respond to oleic acid induction. And it has different response intensities for different oleic acid concentrations.

Usage and Biology

The FadR protein expressed by the FadR gene can bind to the operator FadO following the FadBA promoter, which inhibits the initiation of FadBA. However, oleoyl CoA metabolized from oleic acid can relieve this inhibition and promote the expression of FadBA promoter. This allows it to be induced by oleic acid for expression. In practical applications, we can change the number and position of FadO operators to change their response threshold to oleic acid to achieve more precise regulatory effects.


Characterization

In order to characterize the oleic acid inducer, we added mRFP after the oleic acid inducer for characterization, and we validated the response of the oleic acid inducer to gradient oleic acid by inducing the initiation of the oleic acid promoter under both aerobic and anaerobic conditions, as well as the operability of the part under anaerobic conditions. For detailed information, please refer to BBa_K4788008

Characterization results

To verify the effect of oleic acid inducer, we added mRFP gene after the oleic acid inducer gene to construct a reporter gene. We cultured engineering bacteria under two different environmental conditions, anaerobic and aerobic, and under different oleic acid induction conditions, and detected the expression effect of oleic acid inducers through normalized fluorescence signal intensity. As shown in Figure 1, the oleic acid inducer has a good response to gradient oleic acid, especially in the absence of oxygen, showing significant differences from the previous gradient at a 10% oleic acid gradient.

control
Figure1: Gradient oleic acid induced expression intensity of oleic acid starter under both anaerobic and aerobic conditions.

Under the comparison of anaerobic and aerobic conditions, as shown in Figure 2, there was a slight difference in the expression level of the oleic acid inducer gene, but it did not show significant difference.

control
Figure 2: Comparison of the expression intensity of gradient oleic acid induced oleic acid starter under anaerobic and aerobic conditions.

Reference

Ma Y, Zheng X, Lin Y, Zhang L, Yuan Y, Wang H, Winterburn J, Wu F, Wu Q, Ye JW, Chen GQ. Engineering an oleic acid-induced system for Halomonas, E. coli and Pseudomonas. Metab Eng. 2022 Jul;72:325-336. doi: 10.1016/j.ymben.2022.04.003. Epub 2022 May 2. PMID: 35513297.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 764
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]