Difference between revisions of "Part:BBa K5127004"
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==Team: BNDS-China 2024== | ==Team: BNDS-China 2024== | ||
Our project aims to design a system that could sense the excess of IAA, a harmful metabolite to humans (Wei, W. et al., 2023). Within this framework, we used a mutated version of J23119 to constitutively express iacR. iacR loses its repressing function upon binding with IAA, enabling further gene expression. GFP is adopted as the reporter gene, and it will be replaced with IAA degrading genes in the final construct to achieve real-time biodegradation of IAA (Figure 1). | Our project aims to design a system that could sense the excess of IAA, a harmful metabolite to humans (Wei, W. et al., 2023). Within this framework, we used a mutated version of J23119 to constitutively express iacR. iacR loses its repressing function upon binding with IAA, enabling further gene expression. GFP is adopted as the reporter gene, and it will be replaced with IAA degrading genes in the final construct to achieve real-time biodegradation of IAA (Figure 1). | ||
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Figure 1. Plasmid design of pIacR. Created by biorender.com. | Figure 1. Plasmid design of pIacR. Created by biorender.com. | ||
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===Characterization of IacR-mediated IAA biosensor=== | ===Characterization of IacR-mediated IAA biosensor=== | ||
The plasmid containing the biosensor device shown above was synthesized by Genscript. We used whole-cell kinetics to quantitatively test the effectiveness of IAA induction. A gradient of IAA concentration, from 0μM to 100μM, was added into E. coli transformed with pIacR, and the value of fluorescence / ABS600 over time was detected using the microplate reader to represent GFP expression (Figure 2) . | The plasmid containing the biosensor device shown above was synthesized by Genscript. We used whole-cell kinetics to quantitatively test the effectiveness of IAA induction. A gradient of IAA concentration, from 0μM to 100μM, was added into E. coli transformed with pIacR, and the value of fluorescence / ABS600 over time was detected using the microplate reader to represent GFP expression (Figure 2) . | ||
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Figure 2. Kinetics of pIacR over 16.7 hours. Fluorescence / ABS600 was used to represent GFP expression. The darker blue color, the higher IAA concentration. The numbers represent the concentration (in μM) of IAA added. | Figure 2. Kinetics of pIacR over 16.7 hours. Fluorescence / ABS600 was used to represent GFP expression. The darker blue color, the higher IAA concentration. The numbers represent the concentration (in μM) of IAA added. | ||
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After cultured at 37 ℃ for 18 hrs, the fluorescence/ABS 600 values increased as IAA concentration increased. Particularly, the fluorescence/ABS600 value showed a sharp increase from uninduced (0 µM IAA) to a minimal IAA level, which shows the successful design of the IAA detection module with high sensitivity. | After cultured at 37 ℃ for 18 hrs, the fluorescence/ABS 600 values increased as IAA concentration increased. Particularly, the fluorescence/ABS600 value showed a sharp increase from uninduced (0 µM IAA) to a minimal IAA level, which shows the successful design of the IAA detection module with high sensitivity. | ||
Revision as of 06:50, 2 October 2024
iacR
This part is the coding sequence of iacR which is an allosteric repressor responsive to IAA.
Usage and Biology
IacR is a MarR family repressor that is responsive to IAA. IacR binds to the operator sequence in its apo-form and releases the promoter DNA upon IAA binding. We adopted IacR as the regulator to construct the IAA biosensor.
Team: BNDS-China 2024
Our project aims to design a system that could sense the excess of IAA, a harmful metabolite to humans (Wei, W. et al., 2023). Within this framework, we used a mutated version of J23119 to constitutively express iacR. iacR loses its repressing function upon binding with IAA, enabling further gene expression. GFP is adopted as the reporter gene, and it will be replaced with IAA degrading genes in the final construct to achieve real-time biodegradation of IAA (Figure 1).
Figure 1. Plasmid design of pIacR. Created by biorender.com.
Figure 2. Kinetics of pIacR over 16.7 hours. Fluorescence / ABS600 was used to represent GFP expression. The darker blue color, the higher IAA concentration. The numbers represent the concentration (in μM) of IAA added.