Difference between revisions of "Part:BBa K5127006"

 
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This translation unit belonging to the MarR-family of bacterial regulators, iadCDE, is derived from the bacterial genus Variovorax and discovered sufficient for indole-3-acetic acid (IAA) degradation and signal interference by Conway et al. (2022).
  
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===Usage and Biology===
 
===Usage and Biology===
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It is composed of iadC, an annotated ferredoxin subunit, followed by iadD and iadE which are responsible for degrading IAA molecules.
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==Team: BNDS-China 2024==
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Our design aims to express IAA degradation genes in the presence of IAA, at which point the repressor iadR loses its repressive function, separating from the promoting and in turn activating downstream translation of degradation enzymes encoded by iadCDE.
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===Design of the Sequence===
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Although in the original paper, these genes all come behind one RBS, we found that adding an RBS before each gene is more achievable. The adoption of iadR, a transcriptional regulator, upstream of these loci also demonstrates IAA sensory function with high specificity for expressing the IAA degradation protein.
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===Results of iadCDE encoded enzymes expressed in E.coli===
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As shown in both figures, enzymes iadC, iadD, and iadE, weighing 35.7kDa, 49.5kDa and 18.9kDa respectively, showed bands matching these values when induced with IPTG and thus conforming the correct expression of these proteins.
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Fig1. SDS-Page Result of expression of iadC, iadD, and iadE.
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==Applying Salkowski Reagent to quantify degrading effects of IAA==
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To detect the degradation effects of enzymes expressed with iadCDE, we measured their color with the Salkowski Reagent which changes color according to the concentration of IAA molecules. As shown in the degradation curve, strain induced by IPTG demonstrates a much lower level of IAA than the IPTG(-) group and control group, yielding successful results.
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Fig2. Degradation Curve of IAA.
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Revision as of 17:01, 29 September 2024


iadCDE

This translation unit belonging to the MarR-family of bacterial regulators, iadCDE, is derived from the bacterial genus Variovorax and discovered sufficient for indole-3-acetic acid (IAA) degradation and signal interference by Conway et al. (2022).

Usage and Biology

It is composed of iadC, an annotated ferredoxin subunit, followed by iadD and iadE which are responsible for degrading IAA molecules.

Team: BNDS-China 2024

Our design aims to express IAA degradation genes in the presence of IAA, at which point the repressor iadR loses its repressive function, separating from the promoting and in turn activating downstream translation of degradation enzymes encoded by iadCDE.

Design of the Sequence

Although in the original paper, these genes all come behind one RBS, we found that adding an RBS before each gene is more achievable. The adoption of iadR, a transcriptional regulator, upstream of these loci also demonstrates IAA sensory function with high specificity for expressing the IAA degradation protein.

Results of iadCDE encoded enzymes expressed in E.coli

As shown in both figures, enzymes iadC, iadD, and iadE, weighing 35.7kDa, 49.5kDa and 18.9kDa respectively, showed bands matching these values when induced with IPTG and thus conforming the correct expression of these proteins.


Fig1. SDS-Page Result of expression of iadC, iadD, and iadE.

Applying Salkowski Reagent to quantify degrading effects of IAA

To detect the degradation effects of enzymes expressed with iadCDE, we measured their color with the Salkowski Reagent which changes color according to the concentration of IAA molecules. As shown in the degradation curve, strain induced by IPTG demonstrates a much lower level of IAA than the IPTG(-) group and control group, yielding successful results.


Fig2. Degradation Curve of IAA.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 2355
    Illegal PstI site found at 2172
    Illegal PstI site found at 2750
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2355
    Illegal NheI site found at 677
    Illegal PstI site found at 2172
    Illegal PstI site found at 2750
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2355
    Illegal BglII site found at 2364
    Illegal BglII site found at 2416
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 2355
    Illegal PstI site found at 2172
    Illegal PstI site found at 2750
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 2355
    Illegal PstI site found at 2172
    Illegal PstI site found at 2750
    Illegal NgoMIV site found at 623
    Illegal NgoMIV site found at 1614
    Illegal AgeI site found at 387
    Illegal AgeI site found at 1416
    Illegal AgeI site found at 1857
    Illegal AgeI site found at 2280
    Illegal AgeI site found at 2714
  • 1000
    COMPATIBLE WITH RFC[1000]