Part:BBa_K5127027
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
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 2374
Illegal PstI site found at 2191
Illegal PstI site found at 2769 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2374
Illegal BglII site found at 2383
Illegal BglII site found at 2435 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2374
Illegal PstI site found at 2191
Illegal PstI site found at 2769 - 25INCOMPATIBLE 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 - 1000COMPATIBLE WITH RFC[1000]
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