Part:BBa_K5127006
iadCDE
This part is a translational unit for the IAA degradation enzymes iadCDE.
Usage and Biology
The iadCDE genes from V. paradoxus are the minimal set of genes capable of efficiently degrading IAA in a relatively short amount of time. It has also been tested in E. coli and has shown to be effective (Conway et al., 2022). Specifically, iadC encodes a putative ferredoxin subunit; iadD is a putative large terminal subunit of phenylpropionate dioxygenase, and iadE is a putative small subunit of 3-phenylpropionate dioxygenase.
Team: BNDS-China 2024
Our design aims to express IAA degradation enzymes in the presence of IAA, at which point the repressor iacR loses its repressive function, separating from the promoter and in turn trigger the expression of downstream iadCDE gene encoding IAA degradation enzymes.
Characterization of iadCDE
We first characterized iadCDE expression and IAA degradation using pET28a plasmid, with the iadCDE expression induced by IPTG. Because transcription and translation rates are significantly small when we assess the original plasmid from the article using the RBS calculator (Salis, H. M.,2009), we separated the three genes with three stronger RBSs to enhance the gene expression level (Figure 1). We also designed a second version of this degradation module in which the T7 promoter is replaced with the J23119 constitutive promoter for an even higher gene expression level. After confirming the function of the IAA biosensor and degradation devices, we plan to combine them to form a complete circuit.
Figure 1. Plasmid design of PiadCDE. Created by biorender.com.
The iadC, iadD, and iadE were synthesized by Genscript. To verify the successful expression of iadC, iadD, and iadE, we performed SDS-PAGE to the expressed protein in E. coli BL21(DE3) 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 validating the Salkowski reagent, we cultured transformed bacteria together with IAA and made an IAA degradation curve. After IPTG induction, the IAA concentration maintained at a constant level for the uninduced culture and the BL21(DE3) strain without plasmid, while the group with iadCDE expression induced by IPTG showed a decreasing trend of Abs530, which corresponds to a lower IAA level (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.
Verification of J23119-iadCDE
To optimize the IAA degradation efficiency, we constructed a second plasmid that replaces the inducible T7 promoter with the constitutive promoter J23119. This modification ensures continuous expression of the IAA degradation pathway, potentially enhancing its overall effectiveness.
Figure 5. Plasmid design of J23119-iadCDE. Created by biorender.com.
Figure 6. The agarose gel electrophoresis results of the PCR products of J23119-iadCDE construction. A, materials to construct J23119-iadCDE. B, Golden gate assembly result of J23119-iadCDE construction. The band at 7947bp in (B) indicated the success in plasmid construction.
We require additional time to further characterize and evaluate the degradation function to ensure optimal performance and reliability in our system.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 2355
Illegal PstI site found at 2172
Illegal PstI site found at 2750 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2355
Illegal BglII site found at 2364
Illegal BglII site found at 2416 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2355
Illegal PstI site found at 2172
Illegal PstI site found at 2750 - 25INCOMPATIBLE 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 - 1000COMPATIBLE WITH RFC[1000]
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