Part:BBa_K5127013

Butyrate sensor with NOT gate (pPcha-ci-pLam-GFP)

This composite part conbines the pPcha-cI system with pLam-cI NOT gate to reverse the logic in expressing the downstream genes, in which GFP downstream will be inhibit under the existence of butyrate for the characterizaton.

Team: BNDS-China 2024

The leucine-responsive regulatory protein Lrp was constitutively expressed as driven by J23101. Then, Lrp activates the expression of cI repressor in the presence of butyrate, and the cI protein inhibits promoter pLam activity (Figure 1). The pCI-Lam, PpchA, protein CI, and GFP were synthesized by Genscript. The Lrp protein was supported by NMU-China.

Figure 1. Plasmid design of PpchA. Created by biorender.com.

===Characterization of butyrate biosensor using PpchA=== Initially, our plasmid's RBS component, synthesized by Genscript, had an unwanted point mutation. But we did characterization to see whether it could still function well (Figure 2).

Figure 2. Kinetics of PpchA (with RBS point mutation) with multiple butyrate concentrations over 18 hours. Fluorescence / ABS600 was used to represent GFP expression; higher fluorescence represented lower PpchA activity.

The kinetics results of this error-containing plasmid did not achieved a desired performance: the dynamic range of this system was less than 1.5-fold at after added a gradient of butyrate concentration. Therefore, we've tried to fix the point mutation. We used GoldenGate Assembly to construct PpchA-RBS-fixed. PCR and Gel Electrophoresis were performed to verify the success in constructing each component and the plasmid (Figure 3).

Figure 3. The AGE results of the PCR products of PpchA construction. A, materials to construct PpchA. B, Goldengate assembly result of PpchA construction. The band at 5278bp in (B) indicated the success in plasmid construction.

We again used kinetics to quantitatively test the effectiveness of the above design for butyrate detection. A gradient of butyrate concentration was added into E. coli transformed with PpchA, and the value of fluorescence / ABS600 over time was detected using the plate-reader to represent GFP expression levels (Figure 4).

Figure 4. Kinetics of PpchA (RBS fixed) with multiple butyrate concentrations over 16.7 hours. Fluorescence / ABS600 was used to represent GFP expression; higher fluorescence represented lower PpchA activity.

The overall performance of the PpchA-Lrp sensor is sufficient to demonstrate its ability to detect and respond to the presence of butyrate accurately at most butyrate concentrations. However, the sensor exhibits low sensitivity in distinguishing between small variations in butyrate levels; furthermore, the dynamic range of this detection system was only about 2-fold. This is likely attributed to the inherent limitation of this system, as a similar noisy butyrate induction pattern of PpchA-Lrp has been previously reported in literature (Serebrinsky-Duek et al., 2023). To address those limitations, we developed an alternative sensor, pHdpH (BBa_K5127014), for further investigation.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 970
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 239
  
• 1000
  COMPATIBLE WITH RFC[1000]