Part:BBa_K855008

AHL-inducible Production of pvdQ (K855005)

This part requires the external presence acyl homoserine lactones to stimulate the production of PVDQ, but PVDQ produced will degrade the AHL molecules, it is a dynamic system that maintains the AHL concentration at certain level.

Characterization by BS_United_China 2021

See detail in part BBa_K3882002 PVDQ has been known by us and the University of Hong Kong in 2012 as the degrading enzyme of AHL (the most important quorum sensing molecule of Gram-negative bacteria). But in the part we designed this year, we connected the PVDQ protein and eGFP protein with three HA protein linkers, and added two pairs of 6 his tags. Our design is more intuitive and easier to extract PVDQ protein. And we also used the T7 promoter to increase protein expression.

Our ultimate goal in designing this part is to apply the PVDQ protein to daily life. We also designed a plasmid that can detect the concentration of AHL in food. See details in BBa_K3882001 After connecting the two parts we designed, we can detect the growth of bacteria in fresh food and secrete PVDQ protein in time to prevent the flora from releasing toxic substances.

The experiments conducted by our team in the laboratory in 2021 proved:
1. PVDQ protein can inhibit the growth of bacteria (Pseudomonas aeruginosa)

Figure1:Growth curve of the P. aeruginosa.

It has been speculated that the weakening of PVDQ inhibitory effect after 9 hours is due to protein denaturation, decomposition and inactivation under a constant temperature culture environment of 37 degrees Celsius.

2. PVDQ-eGFP was successfully extracted by our non-denaturing elution method and can be stored at -40 degrees Celsius without inactivation.

Figure2:The PVDQ-eGFP produced by BBa_K3882002 induced by IPTG was observed under a fluorescence microscope.

3.IPTG can activate the T7 promoter to initiate the expression of pvdq-gfp gene. As shown in the figure 2, the E. coli are producing the PVDQ-GFP. The growth of BL21 E. coli inserted into our plasmid is not significantly different from that of normal E. coli
4. PVDQ-eGFP has not been decomposed after 30 minutes of treatment in a simulated gastric acid environment. As shown in the figure 3, the PVDQ-GFP can resistant to the low pH environment. Thus, the fusion design of PVDQ-GFP parts can have a long time funciton in the reaction buffer.
5. PVDQ-GFP can not only prevent the growth of P. aeruginosa on the LB plate (Figure 4) but also can protect the shrimp and fish from the contamination of P. aeruginosa (Figure 5).

Figure3: The degradation of PVDQ-GFP (group1 is control group, while group2 is the group reacting with pH=0.61 acetic acid, and group3 is the group reacting with pH=2.14 hydrochloric acid).

6. PVDQ-GFP can not only prevent the growth of P. aeruginosa on the LB plate (Figure 4) but also can protect the shrimp and fish from the contamination ofP. aeruginosa (Figure 5).

Figure4: Statistic analysis of PVDQ-GFP in inhibiting the growth of P. aeruginosa.

Figure5: The protective effect on the food storage using PVDQ-GFP.

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 2284
  Illegal XhoI site found at 2682
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1119
  Illegal NgoMIV site found at 1858
  Illegal NgoMIV site found at 2260
  Illegal NgoMIV site found at 2417
  Illegal NgoMIV site found at 2644
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1005
  Illegal SapI.rc site found at 3014