Intermediate

Part:BBa_K855000

Designed by: Akansha Mahavir Shah, Ma Tsz Shan, Nelson Chu   Group: iGEM12_HKU_HongKong   (2012-09-20)
Revision as of 07:27, 6 October 2012 by ShannonMa (Talk | contribs) (→‎Usage and Biology)

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.


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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