Difference between revisions of "Part:BBa K875005:Experience"
Drikicmarija (Talk | contribs) |
Drikicmarija (Talk | contribs) (→Applications of BBa_K875005</strong) |
||
| Line 11: | Line 11: | ||
<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> | ||
<p style="text-align: justify;"><img style="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></strong></span></p> | <p style="text-align: justify;"><img style="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></strong></span></p> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
<p style="text-align: justify;"><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 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></p> | <p style="text-align: justify;"><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 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></p> | ||
<p style="text-align: justify;"><span style="font-size: 8pt;"></span>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. <br /><span style="font-size: 8pt;"><br /> Reference: <br /> 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. <br /> 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 <br /> <br /></span></p> | <p style="text-align: justify;"><span style="font-size: 8pt;"></span>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. <br /><span style="font-size: 8pt;"><br /> Reference: <br /> 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. <br /> 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 <br /> <br /></span></p> | ||
Revision as of 07:43, 26 September 2012
This experience page is provided so that any user may enter their experience using this part.
Please enter
how you used this part and how it worked out.
Applications of BBa_K875005</strong
===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 style="float: left;" alt="omPasip" src="
" 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
User Reviews
UNIQ3ba4460f7ef2b5b2-partinfo-00000000-QINU UNIQ3ba4460f7ef2b5b2-partinfo-00000001-QINU