Difference between revisions of "Part:BBa K875006:Experience"

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The construct was tested in E.coli W3110 strain which was previously transformed with p-REP 4 coding for the T5 Lac Repressor. The recombinant bacterial colonies were induced at O.D.= 0.4 (2x102 bacterial cells/ml) with IPTG (1mM) at 37°C in shaker. 2ml of bacterial colture was centrifuged and the pellet was resuspended in 200μl of lysis buffer. The samples were then sonificated and boiled for 5 min at 95°C. 10μl of lysates of induced, non-induced and non-trasformed bacterial cultures were resolved on SDS-PAGE. The expression of fusion protein scFv 54.6-His was tested by Western blotting with anti-6HIS antibodies (Fig. 1).  
 
The construct was tested in E.coli W3110 strain which was previously transformed with p-REP 4 coding for the T5 Lac Repressor. The recombinant bacterial colonies were induced at O.D.= 0.4 (2x102 bacterial cells/ml) with IPTG (1mM) at 37°C in shaker. 2ml of bacterial colture was centrifuged and the pellet was resuspended in 200μl of lysis buffer. The samples were then sonificated and boiled for 5 min at 95°C. 10μl of lysates of induced, non-induced and non-trasformed bacterial cultures were resolved on SDS-PAGE. The expression of fusion protein scFv 54.6-His was tested by Western blotting with anti-6HIS antibodies (Fig. 1).  
  
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[[Image:Trieste_IMG_WB_pelB_scFv|center|500px]]
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[[Image:Trieste_IMG_WB_pelB_scFv.png|center|500px]]
  
 
FIG.1 Expression of scFv 54.6 cloned in fusion with the pelB leader sequence. Western blots of lysates (C= with pelB-scFv 54.6; WC= without pelB-scFv 54.6) of E.coli HB2151 bacterial strain expressing the recombinant protein scFv 54.6. The blot was reacted with the monoclonal F24-796 anti-6XHIS antibody.
 
FIG.1 Expression of scFv 54.6 cloned in fusion with the pelB leader sequence. Western blots of lysates (C= with pelB-scFv 54.6; WC= without pelB-scFv 54.6) of E.coli HB2151 bacterial strain expressing the recombinant protein scFv 54.6. The blot was reacted with the monoclonal F24-796 anti-6XHIS antibody.

Revision as of 08:38, 26 September 2012

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Applications of BBa_K875006

The construct was tested in E.coli W3110 strain which was previously transformed with p-REP 4 coding for the T5 Lac Repressor. The recombinant bacterial colonies were induced at O.D.= 0.4 (2x102 bacterial cells/ml) with IPTG (1mM) at 37°C in shaker. 2ml of bacterial colture was centrifuged and the pellet was resuspended in 200μl of lysis buffer. The samples were then sonificated and boiled for 5 min at 95°C. 10μl of lysates of induced, non-induced and non-trasformed bacterial cultures were resolved on SDS-PAGE. The expression of fusion protein scFv 54.6-His was tested by Western blotting with anti-6HIS antibodies (Fig. 1).

Trieste IMG WB pelB scFv.png

FIG.1 Expression of scFv 54.6 cloned in fusion with the pelB leader sequence. Western blots of lysates (C= with pelB-scFv 54.6; WC= without pelB-scFv 54.6) of E.coli HB2151 bacterial strain expressing the recombinant protein scFv 54.6. The blot was reacted with the monoclonal F24-796 anti-6XHIS antibody.

Western blot with anti-6HIS antibodies showed the band corresponding to scFv 54.6 at the expected position in the IPTG-iduced sample. In the non-induced sample, a weaker signal is also detected suggesting that the promoter is leaky. Aspecific signals are visible also. Some of them are due to proteins partially degraded.

Reference: 1. “Recombinant norovirus-specific scFv inhibit virus-like particle binding to cellular ligands” K. Ettayebi and M. E. Hardy. Published: 31 January 2008 in Virology Journal 2008, 5:21

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