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Team:Trieste/parts/5
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<div class="box_contenuti"> | <div class="box_contenuti"> | ||
<h2>Description </h2> | <h2>Description </h2> | ||
| - | This construct is designed for the expression of an already | + | <br/> |
| + | <p> | ||
| + | <center><img src="https://static.igem.org/mediawiki/2012/4/44/Treieste_ompA_SIP.png" alt="OmpA-SIP-Circuit"></center> | ||
| + | </p> | ||
| + | <br/> | ||
| + | This construct is designed for the expression of an already described engineered antinorovirus (NoV) monoclonal antibody (mAb 54.6) in fusion with LPP-OmpA. | ||
The antibody is expressed in a small immuno protein (SIP) format containing light (VL) and heavy (VH) variable domains separeted by a flexible peptide linker and constant domain (CH3) of heavy chain of human immunoglobulin A (IgA). It has already been reported that the scFv 54.6 (which compones SIP) binds a native recombinant NoV particles (VLPs) and inhibits VLP interaction with cells. Alpha isotype CH3 domain homodimerizes confering bivalent binding properties to the antibody. | The antibody is expressed in a small immuno protein (SIP) format containing light (VL) and heavy (VH) variable domains separeted by a flexible peptide linker and constant domain (CH3) of heavy chain of human immunoglobulin A (IgA). It has already been reported that the scFv 54.6 (which compones SIP) binds a native recombinant NoV particles (VLPs) and inhibits VLP interaction with cells. Alpha isotype CH3 domain homodimerizes confering bivalent binding properties to the antibody. | ||
| - | LPP-OmpA functions as a leader sequence and an anchor to display the scFv ot the bacterial surface.< | + | LPP-OmpA functions as a leader sequence and an anchor to display the scFv ot the bacterial surface. |
| - | + | <br/> | |
| + | <br/> | ||
The construct consistes of T5 Lac Operator (Bba_K875002), ribosomal binding site, LPP-OmpA, scFv 54.6 antinorovirus, Hystidine tag (6HIS), Terminator (B0015). | The construct consistes of T5 Lac Operator (Bba_K875002), ribosomal binding site, LPP-OmpA, scFv 54.6 antinorovirus, Hystidine tag (6HIS), Terminator (B0015). | ||
| - | </br> | + | <br/> |
| - | < | + | <br/> |
| + | The expression of chimeric protein LPP-OmpA-SIP 54.6-6HIS is regulated by T5 Lac Operator (Bba_K875002). When the promoter T5 Lac O is induced with IPTG (1mM), the fusion protein LPP-OmpA-SIP 54.6-6HIS is expected to be expressed extracellularly on the bacterial surface. LPP-OmpA introduces itself into the outer membrane displaying extracellularly the antibody attached on its C-terminus. | ||
| + | <br/> | ||
| + | <br/> | ||
| + | Molecular Weight: 60 kDa. | ||
| - | </ | + | </p> |
| + | |||
| + | <h2>Assembly</h2> | ||
| + | <p class="fancy"> | ||
| + | </p> | ||
| + | The sequence LPP-OmpA-SIP was obtained by synthesis. | ||
| + | </p> | ||
| + | <p class="fancy"> | ||
<h2>Results</h2> | <h2>Results</h2> | ||
| - | </br> | + | The cloning success has been verified by Colony PCR. (Fig. 1) |
| - | < | + | The construct has been completely sequenced. |
| - | </br> | + | <br/> |
| + | <br/> | ||
| + | <img src="https://static.igem.org/mediawiki/2012/4/42/IMG_ompa_SIP.png" alt="Gel OmpA-SIP" width=650px"/> | ||
| + | <p class="didascalia"><strong>FIG.1 Electrophoresis in gel 1% agarose whit ethidium bromide of T5LacO-LPP-OmpA-SIP-6His-TT (blue box).</strong> Fragment LPP-OmpA-SIP-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 <i>E.coli</i> 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. 2). | ||
| + | <br/> | ||
| + | <br/> | ||
| + | <img src="https://static.igem.org/mediawiki/2012/2/27/OmpaSIP_IMANEW.png" alt="WB OmpA-SIP" width=500px"/> | ||
| + | <p class="didascalia"><strong>FIG. 2. Expression of SIP 54.6 cloned in fusion with the LPP-OmpA leader sequence.</strong> Western blots of lysates of <i>E.coli</i> W3110 bacterial strain expressing the recombinant protein SIP 54.6 (60KDa), induced or non-induced with IPTG. The blot was reacted with the Affinity purified antibody goat anti-Human IgA(α). | ||
| + | </p> | ||
| + | |||
| + | 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. | ||
| + | <br/> | ||
| + | <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/> | ||
<h2>Looking forward</h2> | <h2>Looking forward</h2> | ||
| - | </br> | + | In the next step we will clone the LPP-OmpA-SIP under the constitutive promoter BBa_J23100 and transform it in E.coli Nissle 1917. |
| + | <br/> | ||
| + | <br/> | ||
<h3><a href="http://partsregistry.org/Part:BBa_K875005"target="_blank">Link to the Registry</a></h3> | <h3><a href="http://partsregistry.org/Part:BBa_K875005"target="_blank">Link to the Registry</a></h3> | ||
</br> | </br> | ||
| Line 38: | Line 83: | ||
<li><a href="https://2012.igem.org/Team:Trieste/parts/3">BBa_K875003 - CymR</a></li> | <li><a href="https://2012.igem.org/Team:Trieste/parts/3">BBa_K875003 - CymR</a></li> | ||
<li><a href="https://2012.igem.org/Team:Trieste/parts/4">BBa_K875004 - OmpA scFv</a></li> | <li><a href="https://2012.igem.org/Team:Trieste/parts/4">BBa_K875004 - OmpA scFv</a></li> | ||
| - | <li><a href="https://2012.igem.org/Team:Trieste/parts/5">BBa_K875005 - OmpA SIP</a></li> | + | <li class="select"><a href="https://2012.igem.org/Team:Trieste/parts/5">BBa_K875005 - OmpA SIP</a></li> |
<li><a href="https://2012.igem.org/Team:Trieste/parts/6">BBa_K875006 - PelB scFv</a></li> | <li><a href="https://2012.igem.org/Team:Trieste/parts/6">BBa_K875006 - PelB scFv</a></li> | ||
<li><a href="https://2012.igem.org/Team:Trieste/parts/7">BBa_K875007 - PelB SIP </a></li> | <li><a href="https://2012.igem.org/Team:Trieste/parts/7">BBa_K875007 - PelB SIP </a></li> | ||
Latest revision as of 01:41, 27 September 2012
The Jolly JoCare
a safe probiotic platform for protein expression
BBa_K875005
More
Description
This construct is designed for the expression of an already described engineered antinorovirus (NoV) monoclonal antibody (mAb 54.6) in fusion with LPP-OmpA. The antibody is expressed in a small immuno protein (SIP) format containing light (VL) and heavy (VH) variable domains separeted by a flexible peptide linker and constant domain (CH3) of heavy chain of human immunoglobulin A (IgA). It has already been reported that the scFv 54.6 (which compones SIP) binds a native recombinant NoV particles (VLPs) and inhibits VLP interaction with cells. Alpha isotype CH3 domain homodimerizes confering bivalent binding properties to the antibody. LPP-OmpA functions as a leader sequence and an anchor to display the scFv ot the bacterial surface.
The construct consistes of T5 Lac Operator (Bba_K875002), ribosomal binding site, LPP-OmpA, scFv 54.6 antinorovirus, Hystidine tag (6HIS), Terminator (B0015).
The expression of chimeric protein LPP-OmpA-SIP 54.6-6HIS is regulated by T5 Lac Operator (Bba_K875002). When the promoter T5 Lac O is induced with IPTG (1mM), the fusion protein LPP-OmpA-SIP 54.6-6HIS is expected to be expressed extracellularly on the bacterial surface. LPP-OmpA introduces itself into the outer membrane displaying extracellularly the antibody attached on its C-terminus.
Molecular Weight: 60 kDa.
Assembly
The sequence LPP-OmpA-SIP was obtained by synthesis.
Results
The cloning success has been verified by Colony PCR. (Fig. 1) The construct has been completely sequenced.
FIG.1 Electrophoresis in gel 1% agarose whit ethidium bromide of T5LacO-LPP-OmpA-SIP-6His-TT (blue box). Fragment LPP-OmpA-SIP-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 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. 2).
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 (60KDa), 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 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
Looking forward
In the next step we will clone the LPP-OmpA-SIP under the constitutive promoter BBa_J23100 and transform it in E.coli Nissle 1917.Link to the Registry
