Part:BBa_K2560304

3-HP Sensor. This part is a PhytoBrick variant

This part contains the promoter as well as the coding sequence for the activator HdpR in the 5´region of the part. It also contains the promotor P_HdpR which is regulated by HdpR. This promoter is oriented in reverse to the HdpR codon, facing out of the 3`end of the part. When HdpR binds 3-Hydroxypropionate it changes its conformation, enabling it to bind to P_HdpR and activate transcription of the gene controlled by it.

Usage and Biology

• Inducer: 3-Hydroxypropionate
• 3-Hydroxypropionate is harmless. For induction, a concentration between 1 and 20 mM can be used.
• The system stems from the S1 safety strain of P. putida KT2440.
• We designed this biobrick in order to get a system with a usable readout for the intracellular 3-Hydroxypropionate concentration.
• Can with be combined with a several reporter genes. We had the best results using the LUX operon.

Figure 1: HIER TEXT
this is a schematic, illustrating the working mechanism of the 3HPA sensor

Team: BNDS-China 2024

We firstly characterized the butyrate induction by constitutively expressing HpdR driven by Plpp1 promoter and placing GFP downstream the previously optimized pHpdH promoter with deletion of a palindromic sequence (Figure 1) (Wang et al., 2021).

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

Build

The hpdR, phpdH, and phpdR were synthesized from Gensript. We used Golden Gate Assembly to construct pHdpH. PCR and Gel Electrophoresis were performed to verify the success in constructing the fragment and backbone of the overall pHdpH plasmid (Figure 2).

Figure 2. The AGE result of the PCR products of pHdpH construction. A, materials to construct pHdpH. B, golden gate assembly result of pHdpH construction. The band at 4555bp in (B) indicated the success in plasmid construction.

Result

We used kinetics to test the effectiveness of this butyrate detection system quantitatively. A gradient of butyrate concentration was added into E. coli transformed with the plasmid, and the value of fluorescence / ABS600 over time was detected using plate-reader to represent GFP expression (Figure 3).

Figure 3. Kinetics of GFP expression over 16.7 hours. Fluorescence / ABS was used to represent GFP expression. Black, no butyrate added. Purple, 0.3mM butyrate. Blue, 0.6mM butyrate. Cyan, 1.2mM butyrate. Green, 2.4mM butyrate. Yellow, 4.8mM butyrate. Orange, 9.8mM butyrate. Red, 20mM butyrate. Pink, 40mM butyrate. Gray, 86mM butyrate.

When the butyrate concentration was below 20mM, our system showed the desired trend, as fluorescence increases proportionally with butyrate concentration. Above this point, however, the system failed to distinguish between 20mM, 40mM, and 86mM butyrate. This might be because 20mM butyrate reached the saturation concentration of this biosensor. Since our goal was to detect the shortage of butyrate in the gut system, the sensitivity of our biosensor at low butyrate concentrations is sufficient.

Characterization of butyrate biosensor using pHpdH-CI

To integrate the butyrate biosensor into our ultimate project goal, we designed the CI gene into the pHpdH sensing system plasmid. In this system, when butyrate levels are low, the output of GFP will be higher (Figure 4). This design enables us to monitor and respond to butyrate concentration effectively within our system.

Figure 3. The AGE results of the PCR products of pHpdH-CI construction. The materials used to construct pHpdH-CI.

We require additional time to further characterize and evaluate the degradation function to ensure optimal performance and reliability in our system.

Reference

Wang, J., Zhang, R., Zhang, J., Gong, X., Jiang, T., Sun, X., Shen, X., Wang, J., Yuan, Q., & Yan, Y. (2021). Tunable hybrid carbon metabolism coordination for the carbon-efficient biosynthesis of 1,3-butanediol inEscherichia coli. Green Chemistry, 23(21), 8694–8706. https://doi.org/10.1039/d1gc02867g

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1108
  Illegal NotI site found at 279
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 263
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]