Part:BBa_K5127024
iacR-pIacR-GFP
This composite part combines the iacR transcriptional regulator and the GFP after the piacR to enable the characterization of the system.
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.
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).
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.
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 732
- 1000COMPATIBLE WITH RFC[1000]
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