Revision as of 23:36, 26 September 2012

Team Trieste iGEM 2012

BBa_K875006

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Description

This construct is designed for the expression of secreted, already described engineered antinorovirus (NoV) monoclonal antibody (mAb 54.6).
The antibody is expressed in a single chain fragment variable (scFv) format containing light (VL) and heavy (VH) variable domains separated by a flexible peptide linker. It has already been reported that the scFv 54.6 binds a native recombinant NoV particles (VLPs) and inhibits VLP interaction with cells. PelB is a leader sequence often used to produce secreted proteins.

The construct consist of T5 Lac Operator (Bba_K875002), ribosomal binding site, PelB-scFv 54.6 antinorovirus, HIstidine tag (6HIS), Terminator (B0015).

The expression of the protein PelB-scFv 54.6-HIS is regulated by T5 Lac Operator (Bba_K875002). When the promoter T5 Lac O is induced with IPTG (1mM), scFv 54.6 is secreted. PelB drives the protein into the periplasmic space where is clived off. The resulting protein then can be released into extracellular space by passing through porins channels.

Molecular Weight: 29,25 kDa.

Assembly

The sequence PelB-scFv is obtained by PelB-SIP construct after cut off the region CH3 from SIP. Then through cloning we created the final construct.

Results

The cloning success has been verified by Colony PCR. (Fig. 1) The construct has been completely sequenced.

pelBscFv

FIG.1 Electrophoresis in gel 1% agarose whit ethidium bromide of T5LacO-pelB-scFv-6His-TT (blu selection). Fragment pelB-scFv-6His-TT, previously double digested with XbaI/PstI, was then cloned downstream the T5LacOperator in plasmid pSB1C3 double digested with SpeI/PstI.

The construct was tested in E.coli W3110 strain which was previously transformed with p-REP 4 encoding for the Lac Repressor. The recombinant bacterial colonies were induced at O.D.= 0.4 (2x108 bacterial cells/ml) with IPTG (1mM) at 37°C in shaker. 2ml of bacterial colture 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 scFv 54.6-His was tested by Western blotting with anti-6HIS antibodies (Fig. 2).

pelbScFv2

FIG.2 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 (29,25KDa). 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

Looking forward

In the next step we will clone PelB-scFv under the constitutive promoter BBa_J23100 and transform it in E.coli Nissle 1917

Contact us

For other information, write to:

igem2012@gmail.com

@@ Line 44: / Line 44: @@
 <p class="didascalia"> <strong> FIG.1 Electrophoresis in gel 1% agarose whit ethidium bromide of T5LacO-pelB-scFv-6His-TT (blu selection).</strong> Fragment pelB-scFv-6His-TT, previously double digested with XbaI/PstI, was then cloned downstream the T5LacOperator in plasmid pSB1C3 double digested with SpeI/PstI.</p>
-The construct was tested in E.coli W3110 strain which was previously transformed with p-REP 4 encoding for the Lac Repressor. The recombinant bacterial colonies were induced at O.D.= 0.4 (2x108 bacterial cells/ml) with IPTG (1mM) at 37°C in shaker. 2ml of bacterial colture 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 scFv 54.6-His was tested by Western blotting with anti-6HIS antibodies (Fig. 2).
+The construct was tested in <i>E.coli</i> W3110 strain which was previously transformed with p-REP 4 encoding for the Lac Repressor. The recombinant bacterial colonies were induced at O.D.= 0.4 (2x108 bacterial cells/ml) with IPTG (1mM) at 37°C in shaker. 2ml of bacterial colture 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 scFv 54.6-His was tested by Western blotting with anti-6HIS antibodies (Fig. 2).
 <br/><br/>
 <td><img src="https://static.igem.org/mediawiki/2012/1/16/TRIESTE_SCFV_MG.png" alt="pelbScFv2" width="700px"/></td>
 <br/>
-<p class="didascalia"> <strong> FIG.2 Expression of scFv 54.6 cloned in fusion with the pelB leader sequence. </strong> 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 (29,25KDa). The blot was reacted with the monoclonal F24-796 anti-6XHIS antibody.</p><br/>
+<p class="didascalia"> <strong> FIG.2 Expression of scFv 54.6 cloned in fusion with the pelB leader sequence. </strong> Western blots of lysates (C= with pelB-scFv 54.6; WC= without pelB-scFv 54.6) of <i>E.coli</i> HB2151 bacterial strain expressing the recombinant protein scFv 54.6 (29,25KDa). The blot was reacted with the monoclonal F24-796 anti-6XHIS antibody.</p><br/>
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