Difference between revisions of "Part:BBa K5127006"
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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. | 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== | ==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. | 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=== | ===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. | 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=== | ||
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. | 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. | Fig1. SDS-Page Result of expression of iadC, iadD, and iadE. | ||
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| + | ===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. | 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. | ||
Revision as of 17:58, 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
- 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]