Composite

Part:BBa_K855008

Designed by: Ma Tsz Shan   Group: iGEM12_HKU_HongKong   (2012-10-05)
Revision as of 02:37, 21 October 2021 by Verity (Talk | contribs)


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.

Part2 sequence.png

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.

<b>The PVDQ-eGFP produced by BBa_K3882002 induced by IPTG was observed under a fluorescence microscope

3. IPTG can activate the T7 promoter to enhance the expression of its downstream genes
4. The growth of BL21 E. coli inserted into our plasmid is not significantly different from that of normal E. coli
5. PVDQ-eGFP has not been decomposed after 30 minutes of treatment in a simulated gastric acid environment

Figure3: Add PVDQ to a mixed solution of 3% pH=0.61 hydrochloric acid and 17% pH=2.14 acetic acid at 37 degrees Celsius to decompose and react for half an hour. The picture shows the result of SDS-PAGE protein gel electrophoresis after the reaction



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


[edit]
Categories
Parameters
None