Difference between revisions of "Part:BBa K855000:Experience"

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Applications of BBa_K855000

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.

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

User Reviews

UNIQe133721fc962d95b-partinfo-00000000-QINU UNIQe133721fc962d95b-partinfo-00000001-QINU

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 [[Image:western (800x430).jpg]]
-'''Figure 1: Developed X-ray Film of Western Blot Transfer to Evaluate pvdQ Expression　'''''Italic text''　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
+'''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
 ===User Reviews===