Difference between revisions of "Part:BBa K855000"

 
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<partinfo>BBa_K855000 short</partinfo>
 
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===Usage and Biology===
 
===Usage and Biology===
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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.
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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.
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[[Image:western (800x430).jpg]]
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'''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
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C12-AHL Assay
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[[Image:Ahl standard (800x762).jpg]]
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'''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.
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[[Image:Ahl data (800x783).jpg]]
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'''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.
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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.
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Revision as of 07:16, 6 October 2012

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.

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