Part:BBa_K875004:Experience

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_K875004

The construct was tested in E.coli W3110 strain which was previously trasformed with p-REP 4 encoding for the Lac Repressor. The recombinant bacterial culture was induced at O.D.= 0.4 (2x108 bacterial cells/ml) with IPTG (1mM) at 37°C in shaker. 2ml of bacterial culture were centrifuged and the pellet was resuspended in 200μl of lysis buffer. The samples were then sonicated and boiled for 5 min. 10μl of lysates of induced, non-induced and non trasformed bacterial cultures 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).

FIG. 1. Expression of scFv 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 scFv 54.6 (45,19KDa), induced or non-induced with IPTG. The blot was reacted with the monoclonal F24-796 anti-6XHIS antibody.

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 partially degraded protein.

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 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

UNIQ973b121c401bd1cc-partinfo-00000000-QINU UNIQ973b121c401bd1cc-partinfo-00000001-QINU