Intermediate

Part:BBa_K855000

Designed by: Akansha Mahavir Shah, Ma Tsz Shan, Nelson Chu   Group: iGEM12_HKU_HongKong   (2012-09-20)
Revision as of 01:57, 7 October 2021 by Zzywb2005 (Talk | contribs) (→‎Characterization by BS_United_China 2021)

pvdQ-B0015

This part consists of the gene pvdQ isolated from Pseudomonas areuginosa. pvdQ is an acylase that is capable of degrading long-chain acyl homoserine lactone (AHL) molecules with 6 to 12 carbons.

Usage and Biology

BBa_K855000 is tested to be produce function pvdQ which can degrade C12-AHL molecules. BBa_K855000 is inserted into commercial expression vector pET-21(a) whose transcription is under the control of T7 promoter and a modified lac operon system. The recombinant vector is transformed into BL21 expression host cell. After the culture reaches an OD of 0.6, the protein expression is induced using 0.5mM IPTG.

The bacteria harvested is divided into two groups undergo sonication and osmotic shock which give three samples, (1)Sonication Supernatant(2)Sonication Pellet(3)Osmotic Shock Fluid. IPTG induced BL21 without plasmid is used as negative control.

Western (800x430).jpg

Figure 1: Developed X-ray Film of Western Blot Transfer to Evaluate pvdQ Expression　　The pvdQ protein is subject to posttranslational processing resulting in its autocatalytic cleavage into an alpha subunit (18kDa) and a beta subunit (60kDa). As the alpha subunit does not contain the HisTag, its expression is not visible through this method. The positive controls are two HisTag standard proteins of 50 and 80 kDa. The negative control is the IPTG induced BL21 without the plasmid (the whole cell bacterium). As the 60kDa band is absent from the negative control lane but present in the sonication supernatant and pellet, the pvdQ protein was successfully expressed in the commercial vector and exits in both a soluble and insoluble state. Moreover, the osmotic shock fraction that is the periplasmic portion of the cell also exhibits a 60kDa band. This confirms that the signal peptide on pvdQ functions to translocate it to the periplasm of the host cell as well

C12-AHL Assay Ahl standard (800x762).jpg

Figure 2:　　Fresh samples of (1)Sonication supernatant, (2) Sonication Pellet and (3) Whole cells are mixed with 15ug AHL as well as their boiled fractions for 4 hours at 30 degree celsius. Also, each sample in incubated alone without AHL to adjusted their coresponding Background absorbance value.


Ahl data (800x783).jpg

Figure 3：PBS acts at a negative control to monitor the natural degradation of AHL molecules. Boiling of each of the respective samples denatures the proteins and decreases their activity as an AHL acylase. Therefore, compared to the unboiled, intact samples, more AHL remained after 4 hours of incubation in the boiled samples for each graph. A greater degree of AHL degradation was observed for the sonication supernatant than the pellet. This might imply that most of the pvdQ enzyme is present in the soluble form or the activity of pvdQ in the supernatant is greater.


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 No part name specified with partinfo tag. After connecting the two parts we designed, we can detect the growth of food flora 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)
2. pvdQ-eGFP was successfully extracted by our non-denaturing elution method and can be stored at -40 degrees Celsius without inactivation.
3. IPTG can activate the T7 promoter to enhance the expression of its downstream genes
4. The growth of BL21-shaped E. coli inserted into our plasmid is not significantly different from that of normal E. coli



Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1489
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1489
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1196
    Illegal XhoI site found at 1594
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1489
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1489
    Illegal NgoMIV site found at 31
    Illegal NgoMIV site found at 770
    Illegal NgoMIV site found at 1172
    Illegal NgoMIV site found at 1329
    Illegal NgoMIV site found at 1556
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1926


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