Difference between revisions of "Part:BBa K5127027"
 
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This composite part combines the T7 promotor and transcriptional unit iadCDE.
 
This composite part combines the T7 promotor and transcriptional unit iadCDE.
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==Team: BNDS-China 2024==
 
==Team: BNDS-China 2024==
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===Design===
 
===Design===
 
In the IAA degradation module, we used IPTG to induce the expression of iadC, iadD, and iadE. They function together to degrade IAA (Figure 1).
 
In the IAA degradation module, we used IPTG to induce the expression of iadC, iadD, and iadE. They function together to degrade IAA (Figure 1).
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Figure 1. Plasmid design of PiadCDE. Created by biorender.com.
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<i>Figure 1. Plasmid design of PiadCDE. Created by biorender.com.</i>
 
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===Result===
 
===Result===
 
The iadC, iadD, and iadE were synthesized by Genscript. To verify the successful expression of iadC, iadD, and iadE, we performed PAGE to the expressed protein of E. coli transformed with PiadCDE with or without IPTG added. The bands at indicated lengths showed the successful expression of iadCDE (Figure 2).
 
The iadC, iadD, and iadE were synthesized by Genscript. To verify the successful expression of iadC, iadD, and iadE, we performed PAGE to the expressed protein of E. coli transformed with PiadCDE with or without IPTG added. The bands at indicated lengths showed the successful expression of iadCDE (Figure 2).
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<p style="text-align:center;"><img src="https://static.igem.wiki/teams/5127/results/35.jpg" width="400" height="auto"/>
 
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Figure 2. SDS-PAGE result of iadCDE expression. Lane 1, 6.5-200kDa protein ladder. Lane 2-4, PiadCDE with IPTG(+). Lane 5, PiadCDE with IPTG(-). Lane 6, 6.5-200kDa protein ladder. Lane 7-8 PiadCDE with IPTG(-).
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<i>Figure 2. SDS-PAGE result of iadCDE expression. Lane 1, 6.5-200kDa protein ladder. Lane 2-4, PiadCDE with IPTG(+). Lane 5, PiadCDE with IPTG(-). Lane 6, 6.5-200kDa protein ladder. Lane 7-8 PiadCDE with IPTG(-).</i>
 
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To verify the effectiveness of iadCDE degrading IAA, we used the Salkowski reagent to quantitatively detect the amount of IAA (Gordon et al., 1951). First, we constructed an IAA standard curve to show the feasibility of using the reagent to detect IAA. A gradient of IAA was mixed with Salkowski reagent (see our protocols) and waited for 1 hour for color formation. The absorbance at 530 nm was measured to indicate the IAA concentration, which shows an approximately linear relationship and thus validated the effectiveness of the Salkowski reagent in IAA concentration measurement (Figure 3).
 
To verify the effectiveness of iadCDE degrading IAA, we used the Salkowski reagent to quantitatively detect the amount of IAA (Gordon et al., 1951). First, we constructed an IAA standard curve to show the feasibility of using the reagent to detect IAA. A gradient of IAA was mixed with Salkowski reagent (see our protocols) and waited for 1 hour for color formation. The absorbance at 530 nm was measured to indicate the IAA concentration, which shows an approximately linear relationship and thus validated the effectiveness of the Salkowski reagent in IAA concentration measurement (Figure 3).
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<HTML>
 
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<p style="text-align:center;"><img src="https://static.igem.wiki/teams/5127/results/36.jpg" width="400" height="auto"/>
 
<p style="text-align:center;"><img src="https://static.igem.wiki/teams/5127/results/36.jpg" width="400" height="auto"/>
 
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Figure 3. IAA standard curve.  
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<i>Figure 3. IAA standard curve.</i>
 
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After ensuring the usefulness of the reagent, we cultured transformed bacteria together with IAA and made an IAA degradation curve. After induction, the group with PiadCDE but without IPTG showed higher IAA concentration than BL21 WT, while the group with PiadCDE with IPTG added showed a much lower amount of IAA compared to BL21 WT (Figure 4). This result showed our system could successfully degrade IAA by expressing iadCDE.
 
After ensuring the usefulness of the reagent, we cultured transformed bacteria together with IAA and made an IAA degradation curve. After induction, the group with PiadCDE but without IPTG showed higher IAA concentration than BL21 WT, while the group with PiadCDE with IPTG added showed a much lower amount of IAA compared to BL21 WT (Figure 4). This result showed our system could successfully degrade IAA by expressing iadCDE.
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<HTML>
 
<HTML>
  
 
<p style="text-align:center;"><img src="https://static.igem.wiki/teams/5127/results/37.jpg" width="400" height="auto"/>
 
<p style="text-align:center;"><img src="https://static.igem.wiki/teams/5127/results/37.jpg" width="400" height="auto"/>
 
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Figure 4. IAA degradation curve. Black, BL21 WT. Red, E. coli transformed with PiadCDE with IPTG added. Blue, E. coli transformed with PiadCDE without IPTG added.
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<i>Figure 4. IAA degradation curve. Black, BL21 WT. Red, E. coli transformed with PiadCDE with IPTG added. Blue, E. coli transformed with PiadCDE without IPTG added.</i>
 
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Latest revision as of 10:17, 2 October 2024


IPTG-inducible promoter with IAA degradation enzyme (T7-iadCDE）

This composite part combines the T7 promotor and transcriptional unit iadCDE.


Team: BNDS-China 2024

Design

In the IAA degradation module, we used IPTG to induce the expression of iadC, iadD, and iadE. They function together to degrade IAA (Figure 1).


Figure 1. Plasmid design of PiadCDE. Created by biorender.com.


Result

The iadC, iadD, and iadE were synthesized by Genscript. To verify the successful expression of iadC, iadD, and iadE, we performed PAGE to the expressed protein of E. coli transformed with PiadCDE with or without IPTG added. The bands at indicated lengths showed the successful expression of iadCDE (Figure 2).


Figure 2. SDS-PAGE result of iadCDE expression. Lane 1, 6.5-200kDa protein ladder. Lane 2-4, PiadCDE with IPTG(+). Lane 5, PiadCDE with IPTG(-). Lane 6, 6.5-200kDa protein ladder. Lane 7-8 PiadCDE with IPTG(-).


To verify the effectiveness of iadCDE degrading IAA, we used the Salkowski reagent to quantitatively detect the amount of IAA (Gordon et al., 1951). First, we constructed an IAA standard curve to show the feasibility of using the reagent to detect IAA. A gradient of IAA was mixed with Salkowski reagent (see our protocols) and waited for 1 hour for color formation. The absorbance at 530 nm was measured to indicate the IAA concentration, which shows an approximately linear relationship and thus validated the effectiveness of the Salkowski reagent in IAA concentration measurement (Figure 3).


Figure 3. IAA standard curve.


After ensuring the usefulness of the reagent, we cultured transformed bacteria together with IAA and made an IAA degradation curve. After induction, the group with PiadCDE but without IPTG showed higher IAA concentration than BL21 WT, while the group with PiadCDE with IPTG added showed a much lower amount of IAA compared to BL21 WT (Figure 4). This result showed our system could successfully degrade IAA by expressing iadCDE.


Figure 4. IAA degradation curve. Black, BL21 WT. Red, E. coli transformed with PiadCDE with IPTG added. Blue, E. coli transformed with PiadCDE without IPTG added.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 2374
    Illegal PstI site found at 2191
    Illegal PstI site found at 2769
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2374
    Illegal NheI site found at 696
    Illegal PstI site found at 2191
    Illegal PstI site found at 2769
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2374
    Illegal BglII site found at 2383
    Illegal BglII site found at 2435
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 2374
    Illegal PstI site found at 2191
    Illegal PstI site found at 2769
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 2374
    Illegal PstI site found at 2191
    Illegal PstI site found at 2769
    Illegal NgoMIV site found at 642
    Illegal NgoMIV site found at 1633
    Illegal AgeI site found at 406
    Illegal AgeI site found at 1435
    Illegal AgeI site found at 1876
    Illegal AgeI site found at 2299
    Illegal AgeI site found at 2733
  • 1000
    COMPATIBLE WITH RFC[1000]