Difference between revisions of "Team:UCopenhagen/Gene Construct Design"

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<h1> Design of Gene Construct</h1>
 
<h1> Design of Gene Construct</h1>
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<h2>1. Expression plasmid without chaperone genes</h2>
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This plasmid consists of signal-tagged reporter gene controlled by ara operon, which can be activated by adding arabinose to the culture media. The T3SS signal is N-terminal 20 amino acid sequences from <em>E. coli</em> effector proteins Map. Those signals are linked with the reporter protein by a glycine-serine linker peptide. 6X-His tag is commonly used as a purification tag, but in this case, it can be used as an epitope tag for western blot.
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<p id="gdcalert1" ><span style="color: red; font-weight: bold">>>>>>  gd2md-html alert: inline image link here (to images/Plasmid-construction0.png). Store image on your image server and adjust path/filename if necessary. </span><br>(<a href="#">Back to top</a>)(<a href="#gdcalert2">Next alert</a>)<br><span style="color: red; font-weight: bold">>>>>> </span></p>
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<img src="images/Plasmid-construction0.png" width="" alt="alt_text" title="image_tooltip">
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How to make it?
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<p id="gdcalert2" ><span style="color: red; font-weight: bold">>>>>>  gd2md-html alert: inline image link here (to images/Plasmid-construction1.png). Store image on your image server and adjust path/filename if necessary. </span><br>(<a href="#">Back to top</a>)(<a href="#gdcalert3">Next alert</a>)<br><span style="color: red; font-weight: bold">>>>>> </span></p>
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<img src="images/Plasmid-construction1.png" width="" alt="alt_text" title="image_tooltip">
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<h2>2. Expression plasmid with chaperone genes</h2>
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<p>
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This plasmid consists of signal-tagged reporter gene controlled by ara operon, which can be activated by adding arabinose to the culture media. It also express CesT and CesF effector chaperone operon under a constitutive promoter pCat. Those chaperones assist natural effector protein transport. It has been excluded from the modified LEE region in SIEC strain, but the author put it in the substrate plasmid instead.
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<p id="gdcalert3" ><span style="color: red; font-weight: bold">>>>>>  gd2md-html alert: inline image link here (to images/Plasmid-construction2.png). Store image on your image server and adjust path/filename if necessary. </span><br>(<a href="#">Back to top</a>)(<a href="#gdcalert4">Next alert</a>)<br><span style="color: red; font-weight: bold">>>>>> </span></p>
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<img src="images/Plasmid-construction2.png" width="" alt="alt_text" title="image_tooltip">
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<span style="text-decoration:underline;">How to make it?</span>
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    a.      Construct pBAD vector with CesF/CesT chaperone.
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<p id="gdcalert4" ><span style="color: red; font-weight: bold">>>>>>  gd2md-html alert: inline image link here (to images/Plasmid-construction3.png). Store image on your image server and adjust path/filename if necessary. </span><br>(<a href="#">Back to top</a>)(<a href="#gdcalert5">Next alert</a>)<br><span style="color: red; font-weight: bold">>>>>> </span></p>
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<img src="images/Plasmid-construction3.png" width="" alt="alt_text" title="image_tooltip">
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b. Insert reporter genes with Map20 signal into pBAD-Chaperone plasmid
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<p id="gdcalert5" ><span style="color: red; font-weight: bold">>>>>>  gd2md-html alert: inline image link here (to images/Plasmid-construction4.png). Store image on your image server and adjust path/filename if necessary. </span><br>(<a href="#">Back to top</a>)(<a href="#gdcalert6">Next alert</a>)<br><span style="color: red; font-weight: bold">>>>>> </span></p>
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<img src="images/Plasmid-construction4.png" width="" alt="alt_text" title="image_tooltip">
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3. EspD Translocon expression plasmid
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This plasmid encodes 6His-tagged EspD translocon under pTac, which can be induced by IPTG. The EspD translocon protein are self-assembled hydrophobic membrane protein that sufficient for membrane penetration and pore formation. This translocon protein are normally secreted along with EspB hydrophilic translocon to penetrate host cell membrane and serve as a docking site for the injectisome. This 6His-tagged EspD can be purified by Ni-NTA column or TALON column. It might be used to pre-assemble a translocon on a target membrane to induce injectisome docking on an artificial membrane.
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<p id="gdcalert6" ><span style="color: red; font-weight: bold">>>>>>  gd2md-html alert: inline image link here (to images/Plasmid-construction5.png). Store image on your image server and adjust path/filename if necessary. </span><br>(<a href="#">Back to top</a>)(<a href="#gdcalert7">Next alert</a>)<br><span style="color: red; font-weight: bold">>>>>> </span></p>
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<img src="images/Plasmid-construction5.png" width="" alt="alt_text" title="image_tooltip">
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How to make it?<strong> </strong>
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<strong> </strong>
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<p id="gdcalert7" ><span style="color: red; font-weight: bold">>>>>>  gd2md-html alert: inline image link here (to images/Plasmid-construction6.png). Store image on your image server and adjust path/filename if necessary. </span><br>(<a href="#">Back to top</a>)(<a href="#gdcalert8">Next alert</a>)<br><span style="color: red; font-weight: bold">>>>>> </span></p>
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<img src="images/Plasmid-construction6.png" width="" alt="alt_text" title="image_tooltip">
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<strong> </strong>
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<strong> </strong>
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<strong>Biobrick plasmid to be submitted to iGEM</strong>
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1. Signal sequence biobricks
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2. 6x His-tagged CesT/CesF Chaperone biobrick
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3. 6x His-tagged EspD translocon biobrick
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<strong> </strong>
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</p>
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</html>
 
</html>
 
{{UCopenhagen/Footer}}
 
{{UCopenhagen/Footer}}

Revision as of 09:46, 17 October 2018

HOME
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Design of Gene Construct

1. Expression plasmid without chaperone genes

This plasmid consists of signal-tagged reporter gene controlled by ara operon, which can be activated by adding arabinose to the culture media. The T3SS signal is N-terminal 20 amino acid sequences from E. coli effector proteins Map. Those signals are linked with the reporter protein by a glycine-serine linker peptide. 6X-His tag is commonly used as a purification tag, but in this case, it can be used as an epitope tag for western blot.

>>>>> gd2md-html alert: inline image link here (to images/Plasmid-construction0.png). Store image on your image server and adjust path/filename if necessary.
(Back to top)(Next alert)
>>>>>

alt_text

How to make it?

>>>>> gd2md-html alert: inline image link here (to images/Plasmid-construction1.png). Store image on your image server and adjust path/filename if necessary.
(Back to top)(Next alert)
>>>>>

alt_text

2. Expression plasmid with chaperone genes

This plasmid consists of signal-tagged reporter gene controlled by ara operon, which can be activated by adding arabinose to the culture media. It also express CesT and CesF effector chaperone operon under a constitutive promoter pCat. Those chaperones assist natural effector protein transport. It has been excluded from the modified LEE region in SIEC strain, but the author put it in the substrate plasmid instead.

>>>>> gd2md-html alert: inline image link here (to images/Plasmid-construction2.png). Store image on your image server and adjust path/filename if necessary.
(Back to top)(Next alert)
>>>>>

alt_text

How to make it?

a. Construct pBAD vector with CesF/CesT chaperone.

>>>>> gd2md-html alert: inline image link here (to images/Plasmid-construction3.png). Store image on your image server and adjust path/filename if necessary.
(Back to top)(Next alert)
>>>>>

alt_text

b. Insert reporter genes with Map20 signal into pBAD-Chaperone plasmid

>>>>> gd2md-html alert: inline image link here (to images/Plasmid-construction4.png). Store image on your image server and adjust path/filename if necessary.
(Back to top)(Next alert)
>>>>>

alt_text

3. EspD Translocon expression plasmid

This plasmid encodes 6His-tagged EspD translocon under pTac, which can be induced by IPTG. The EspD translocon protein are self-assembled hydrophobic membrane protein that sufficient for membrane penetration and pore formation. This translocon protein are normally secreted along with EspB hydrophilic translocon to penetrate host cell membrane and serve as a docking site for the injectisome. This 6His-tagged EspD can be purified by Ni-NTA column or TALON column. It might be used to pre-assemble a translocon on a target membrane to induce injectisome docking on an artificial membrane.

>>>>> gd2md-html alert: inline image link here (to images/Plasmid-construction5.png). Store image on your image server and adjust path/filename if necessary.
(Back to top)(Next alert)
>>>>>

alt_text

How to make it?

>>>>> gd2md-html alert: inline image link here (to images/Plasmid-construction6.png). Store image on your image server and adjust path/filename if necessary.
(Back to top)(Next alert)
>>>>>

alt_text

Biobrick plasmid to be submitted to iGEM

1. Signal sequence biobricks

2. 6x His-tagged CesT/CesF Chaperone biobrick

3. 6x His-tagged EspD translocon biobrick