Difference between revisions of "Part:BBa K875005:Experience"

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<p>===Applications of BBa_K875005===</p>
 
<p>===Applications of BBa_K875005===</p>
 
<p style="text-align: justify;">The construct was tested in E.coli W3110 strain which was previously trasformed with p-REP 4 encoding 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 culture was centrifucated 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 coltures were resolved on SDS-PAGE. The expression of fusion protein LPP-OmpA-scFv 54.6-His was tested by Western blotting with anti-6HIS antibodies (Fig. 1).</p>
 
<p style="text-align: justify;">The construct was tested in E.coli W3110 strain which was previously trasformed with p-REP 4 encoding 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 culture was centrifucated 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 coltures were resolved on SDS-PAGE. The expression of fusion protein LPP-OmpA-scFv 54.6-His was tested by Western blotting with anti-6HIS antibodies (Fig. 1).</p>
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<p style="text-align: justify;"><img style="margin-right: 15px; margin-bottom: 15px; float: left;" alt="omPasip" src="https://static.igem.org/mediawiki/2012/7/70/IMG_WB_ompa_SIP.png" height="142" width="424" /><span style="font-size: 8pt;"><strong>FIG. 2. Expression of SIP 54.6 cloned in fusion with the LPP-OmpA leader sequence.</strong> Western blots of lysates&nbsp;of E.coli W3110 bacterial strain expressing the recombinant protein SIP 54.6, induced or non-induced with IPTG. The blot was reacted with the Affinity purified antibody goat anti-Human IgA(α).</span>
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<img alt="omPasip" src="https://static.igem.org/mediawiki/2012/7/70/IMG_WB_ompa_SIP.png" height="142" width="424" />
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<strong>FIG. 2. Expression of SIP 54.6 cloned in fusion with the LPP-OmpA leader sequence.</strong> Western blots of lysates&nbsp;of E.coli W3110 bacterial strain expressing the recombinant protein SIP 54.6, induced or non-induced with IPTG. The blot was reacted with the Affinity purified antibody goat anti-Human IgA(α).</span>
 
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<p style="text-align: justify;"><span style="font-size: 8pt;">&nbsp;</span>
 
<p style="text-align: justify;"><span style="font-size: 8pt;">&nbsp;</span>

Revision as of 07:37, 26 September 2012

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===Applications of BBa_K875005===

The construct was tested in E.coli W3110 strain which was previously trasformed with p-REP 4 encoding 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 culture was centrifucated 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 coltures were resolved on SDS-PAGE. The expression of fusion protein LPP-OmpA-scFv 54.6-His was tested by Western blotting with anti-6HIS antibodies (Fig. 1).

<img alt="omPasip" src="IMG_WB_ompa_SIP.png" height="142" width="424" /> FIG. 2. Expression of SIP 54.6 cloned in fusion with the LPP-OmpA leader sequence. Western blots of lysates of E.coli W3110 bacterial strain expressing the recombinant protein SIP 54.6, induced or non-induced with IPTG. The blot was reacted with the Affinity purified antibody goat anti-Human IgA(α).

 

 

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

Reference:
1. “Transport and anchoring of 8-lactamase to the external surface of Escherichia coli” J. A. Francisco, C. F. Earhart and G. Georgiou. Proc. Natl. Acad. Sci. USA Vol. 89, pp. 2713-2717, April 1992 Biochemistry.
2. “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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