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Team:Trieste/parts/7
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<div class="box_contenuti"> | <div class="box_contenuti"> | ||
<h2>Description </h2> | <h2>Description </h2> | ||
| - | |||
<p> | <p> | ||
| - | < | + | <div class="centrato"> |
| + | <center><img src="https://static.igem.org/mediawiki/2012/3/35/Trieste_PelB_SiP.png" alt="PelB-SIP-Circuit"></center> | ||
| + | </div> | ||
</p> | </p> | ||
<br/> | <br/> | ||
| - | This construct is designed for the expression of secreted, already described | + | 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 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 makes part of 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. PelB is a leader sequence often used to produce secreted proteins. | 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 makes part of 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. PelB is a leader sequence often used to produce secreted proteins. | ||
<br/> | <br/> | ||
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| - | The expression of protein PelB-SIP 54.6-HIS is regulated by T5 Lac Operator (Bba_K875002). When the | + | The expression of protein PelB-SIP 54.6-HIS is regulated by T5 Lac Operator (Bba_K875002). When the promoter T5 Lac O is induced with IPTG (1mM), SIP 54.6 is expressed. PelB drives the protein into the periplasmic space where is clived off. The resulting protein then can be released into the extracellular space passing through porins channels. |
<br/> | <br/> | ||
Molecular Weigth 44.06 kDa. | Molecular Weigth 44.06 kDa. | ||
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<h2>Assembly</h2> | <h2>Assembly</h2> | ||
| - | |||
The sequence PelB-SIP is obtained by synthesis. Then through cloning we created the final construct. | The sequence PelB-SIP is obtained by synthesis. Then through cloning we created the final construct. | ||
<br/> | <br/> | ||
<br/> | <br/> | ||
<h2>Results</h2> | <h2>Results</h2> | ||
| - | |||
The cloning success has been verified by Colony PCR. (Fig. 1) | The cloning success has been verified by Colony PCR. (Fig. 1) | ||
The construct has been completely sequenced. | The construct has been completely sequenced. | ||
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<td><img src="https://static.igem.org/mediawiki/2012/d/d6/Immagine1.png" alt="" width="450px"/> | <td><img src="https://static.igem.org/mediawiki/2012/d/d6/Immagine1.png" alt="" width="450px"/> | ||
| - | <p class="didascalia"><strong>FIG.1 Electrophoresis in gel 1% agarose whit ethidium bromide of T5LacO-pelB-SIP-6His-TT (blu | + | <p class="didascalia"><strong>FIG.1 Electrophoresis in gel 1% agarose whit ethidium bromide of T5LacO-pelB-SIP-6His-TT (blu box).</strong>Fragment pelB-SIP-6His-TT, previously double digested with XbaI/PstI, was cloned downstream the T5LacOperator in plasmid pSB1C3 double digested with SpeI/PstI. |
</p> | </p> | ||
<br/> | <br/> | ||
| - | The construct was tested in E.coli W3110 strain which was previously trasformed with p-REP 4 | + | The construct was tested in<i> E.coli</i> 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 (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 protein SIP 54.6-His was tested by Western blotting with anti-6HIS antibodies (Fig. 2). |
<br/> | <br/> | ||
<br/> | <br/> | ||
<td> | <td> | ||
<p><img src="https://static.igem.org/mediawiki/2012/d/d5/Trieste-PelB-SIP.png" alt="PelB-SIP" width="650" /> | <p><img src="https://static.igem.org/mediawiki/2012/d/d5/Trieste-PelB-SIP.png" alt="PelB-SIP" width="650" /> | ||
| - | <p class="didascalia"><strong>FIG. 2. Expression of SIP 54.6 cloned in fusion with the pelB leader sequence. </strong>Western blots of lysates (SN=supernatant; P=pellet) 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 monoclonal F24-796 anti-6XHIS antibody. | + | <p class="didascalia"><strong>FIG. 2. Expression of SIP 54.6 cloned in fusion with the pelB leader sequence. </strong>Western blots of lysates (SN=supernatant; P=pellet) of <i>E.coli</i> W3110 bacterial strain expressing the recombinant protein SIP 54.6, induced or non-induced with IPTG. The blot was reacted with the monoclonal F24-796 anti-6XHIS antibody. |
</p> | </p> | ||
<br/> | <br/> | ||
| - | Western blot with anti-6HIS antibodies showed the band corresponding to SIP 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 also visible; some of them are due to | + | Western blot with anti-6HIS antibodies showed the band corresponding to SIP 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 also visible; some of them are due to partially degraded proteins. |
<br/> | <br/> | ||
<br/> | <br/> | ||
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<h2>Looking forward</h2> | <h2>Looking forward</h2> | ||
| + | In the next step we will clone PelB-SIP under the constitutive promoter BBa_J23100 and transform it in E.coli Nissle 1917. | ||
| + | </br> | ||
</br> | </br> | ||
<h3><a href="http://partsregistry.org/Part:BBa_K875007"target="_blank">Link to the Registry</a></h3> | <h3><a href="http://partsregistry.org/Part:BBa_K875007"target="_blank">Link to the Registry</a></h3> | ||
Latest revision as of 01:43, 27 September 2012
The Jolly JoCare
a safe probiotic platform for protein expression
BBa_K875007
More
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 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 makes part of 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. PelB is a leader sequence often used to produce secreted proteins.
The construct consists of T5 Lac Operator (Bba_K875002), ribosomal binding site, PelB-SIP 54.6 antinorovirus, Hystidine tag (6HIS), Terminator (B0015).
The expression of protein PelB-SIP 54.6-HIS is regulated by T5 Lac Operator (Bba_K875002). When the promoter T5 Lac O is induced with IPTG (1mM), SIP 54.6 is expressed. PelB drives the protein into the periplasmic space where is clived off. The resulting protein then can be released into the extracellular space passing through porins channels.
Molecular Weigth 44.06 kDa.
Assembly
The sequence PelB-SIP is obtained by synthesis. 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.
FIG.1 Electrophoresis in gel 1% agarose whit ethidium bromide of T5LacO-pelB-SIP-6His-TT (blu box).Fragment pelB-SIP-6His-TT, previously double digested with XbaI/PstI, was 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 T5 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 protein SIP 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 pelB leader sequence. Western blots of lysates (SN=supernatant; P=pellet) 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 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-iduced 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 proteins.
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-SIP under the constitutive promoter BBa_J23100 and transform it in E.coli Nissle 1917.Link to the Registry
