Team:DTU Denmark/USERprinciple

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The USER fusion assembly standard allows rapid construction of multi-part devices, without some of the drawbacks of the restriction-enzyme based standard biobrick assembly method. The full USER<sup>TM</sup> assembly standard can be found here: (<a href="http://openwetware.org/wiki/The_BioBricks_Foundation:RFC#BBF_RFC_39:_The_USER_cloning_standard" target="_blank">BBF RFC 39</a>). The main advantages of this assembly method is:<br>
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    <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2009.igem.org/Team:DTU_Denmark" >Home</a></font> </td>
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    <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2009.igem.org/Team:DTU_Denmark/team" >The Team</a> </font></td>
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    <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2009.igem.org/Team:DTU_Denmark/project" >The Project</a> </font></td>
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    <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2009.igem.org/Team:DTU_Denmark/parts" >Parts submitted</a> </font></td>
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    <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2009.igem.org/Team:DTU_Denmark/modelling">Modelling</a></font> </td>
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    <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2009.igem.org/Team:DTU_Denmark/notebook" title="Day to day lab activity">Notebook</a>
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  <br>
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  The redoxilator<br>
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  </font>
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</p>
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  <font color="" face="arial" size="2">
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    <a href="https://2009.igem.org/Team:DTU_Denmark/genetic_design" CLASS=leftbar>- Genetic design</a><br>
 +
    <a href="https://2009.igem.org/Team:DTU_Denmark/applications" CLASS=leftbar>- Applications and perspectives</a><br>
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    <a href="https://2009.igem.org/Team:DTU_Denmark/safety" CLASS=leftbar>- Safety considerations</a><br>
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  </font>
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<p> 
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  <font color="#990000" face="arial" size="3">
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    <br>The USER assembly standard<br>
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</p> 
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  <font face="arial" size="2">
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    <a href="https://2009.igem.org/Team:DTU_Denmark/USERprinciple" CLASS=leftbar>- USER fusion of biobricks</a><br>
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  </font>
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<p> 
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  <font color="#990000" face="arial" size="3">
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    <br>USER fusion primer design software<br>
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  </font>
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</p> 
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  <font face="arial" size="2">
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    <a href="https://2009.igem.org/Team:DTU_Denmark/USERprogram" CLASS=leftbar>- Abstract</a><br>
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    <a href="https://2009.igem.org/Team:DTU_Denmark/USERprograminstructions" CLASS=leftbar>- Instructions</a><br>
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    <a href="https://2009.igem.org/Team:DTU_Denmark/USERprogramoutputformat" CLASS=leftbar>- Output format</a><br>
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  </font>
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<p> 
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  <font color="#990000" face="arial" size="3">
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    <br>Experimental results<br>
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  </font>
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</p> 
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  <font face="arial" size="2">
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    <a href="https://2009.igem.org/Team:DTU_Denmark/results" CLASS=leftbar>- Results and discussion</a><br>
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  <font color="#990000" face="arial" size="5">
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  <br>
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  <b>The project</b><br><br>
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<!-- INSERT MAIN TEXT HERE! (formatting: <b>bold</> <i>italic> <h4>header</h4>) -->
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<font size="4"><b>The USER fusion assembly standard</b></font><br><br>
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<p align="justify">
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The USER fusion assembly standard allows rapid construction of multi-part devices, without some of the drawbacks of the restriction-enzyme based standard biobrick assembly method. The full USER assembly standard can be found <a href="http://dl.getdropbox.com/u/912376/BBF_RFC_39.pdf"> here</a>: (<a href="http://openwetware.org/wiki/The_BioBricks_Foundation:RFC#BBF_RFC_39:_The_USER_cloning_standard" target="_blank">BBF RFC 39</a>). The main advantages of this assembly method is:<br>
<br>
<br>
-
1. Standardized method for assembling several BioBricks or components at a time, in contrast to the one at a time" assembly procedure normally used.<br>
+
<b>1.</b> Standardized method for assembling several BioBricks or components at a time, in contrast to the one at a time" assembly procedure normally used.<br>
-
2. Since the method only relies on a single 8 bp recognition restriction enzyme, PacI, all restriction sites are allowed in the biobricks except the TTAATTAA PacI recognition site.<br>
+
<b>2.</b> Since the method relies PCR based assembly, all restriction sites are allowed in the biobricks.<br>
-
3. Eight basepair-overhangs allows ligase-free cloning. With the enclosed protocol <i>E. coli</i> can be transformed with a multipart-construct less than 2 hours after your PCR-reaction has completed.<br>
+
<b>3.</b> Eight basepair-overhangs allows ligase-free cloning. With the enclosed protocol <i>E. coli</i> can be transformed with a multipart-construct less than 2 hours after your PCR-reaction has completed.<br>
-
4. The biobricks are joined without leaving a scar which is ideal for fusing protein domain biobricks.<br>
+
<b>4.</b> The biobricks are joined without leaving a scar which is ideal for fusing protein domain biobricks.<br>
-
5. Insertions of small sequences between biobricks such as a intracellular localization signal, restriction site or flexible linker is possible with the right primer design.<br>
+
<b>5.</b> Insertions of small sequences between biobricks such as a intracellular localization signal, restriction site or flexible linker is possible with the right primer design.<br>
-
6. High fidelity is ensured by using PfuTurbo® C<sub>x</sub> Hotstart DNA polymerase.<br>
+
<b>6.</b> High fidelity is ensured by using PfuTurbo® C<sub>x</sub> Hotstart DNA polymerase.<br>
-
7. By the design of the PCR tails, it can be decided whether the USER cassette should be deleted, copied or moved following insertion.<br>
+
<b>7.</b> By the design of the PCR tails, it can be decided whether the USER cassette should be deleted, copied or moved following insertion.<br>
-
8. Directionality of inserts are supported.<br>
+
<b>8.</b> Directionality of inserts are supported.<br>
<br>
<br>
<br>
<br>
-
<p>
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<p align="justify">
-
<strong>USER fusion of biobricks - how it works</strong><br>
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<font size="3"><b>USER fusion of biobricks - how it works</b></font><br>
We have successfully constructed a USER fusion biobrick (<a href="http://partsregistry.org/Part:BBa_K194003" target="_blank">BBa_K194003</a>), and demonstrated that it works as expected. The biobrick includes a DNA-sequence needed for USER-fusion, which consists of a restriction site and two nicking sites. The entire process of fusing two biobricks are illustrated in the figure, and the same can be done for multiple fragments at once.<br>
We have successfully constructed a USER fusion biobrick (<a href="http://partsregistry.org/Part:BBa_K194003" target="_blank">BBa_K194003</a>), and demonstrated that it works as expected. The biobrick includes a DNA-sequence needed for USER-fusion, which consists of a restriction site and two nicking sites. The entire process of fusing two biobricks are illustrated in the figure, and the same can be done for multiple fragments at once.<br>
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[[Image:User_fusion.png|550px|thumb|center|<b>principle of USER fusion </b>]]
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<p align="justify">
<br>
<br>
<strong>Procedure</strong> (please refer to our Biobrick Assembly Standard BBF RFC 39 for a detailed protocol):<br>
<strong>Procedure</strong> (please refer to our Biobrick Assembly Standard BBF RFC 39 for a detailed protocol):<br>
<br>
<br>
-
1) The USER fusion biobrick plasmid is digested with the restriction enzyme pacI and the nicking enzyme Nt.BbvCI (a nicking enzyme cuts only one strand as illustrated on the figure). This process will linearize the plasmid, and make single stranded overhangs (sticky ends).<br>
+
<b>1.</b> The USER fusion biobrick plasmid is digested with the restriction enzyme pacI and the nicking enzyme Nt.BbvCI (a nicking enzyme cuts only one strand as illustrated on the figure). This process will linearize the plasmid, and make single stranded overhangs (sticky ends).<br>
-
2) PCR amplification is performed on the biobricks intended for the fusion. The primer design is facilitated by our novel USER fusion primer design software made for this iGEM project.<br>
+
<b>2.</b> PCR amplification is performed on the biobricks intended for the fusion. The primer design is facilitated by our novel USER fusion primer design software made for this iGEM project.<br>
-
3) USER<sup>TM</sup> enzyme mix is added. This will remove the uracil always included in the primers, making sticky end overhangs on all biobricks. Because of the matching sticky ends on all biobricks and linearized plamid, the biobricks will self-assemble in the plasmid.<br>
+
<b>3.</b> USER enzyme mix is added. This will remove the uracil always included in the primers, making sticky end overhangs on all biobricks. Because of the matching sticky ends on all biobricks and linearized plamid, the biobricks will self-assemble in the plasmid.<br>
-
Two or more biobricks have been joined with all the advantages mentioned above.
+
Two or more biobricks have been joined with all the advantages mentioned above.<br><br>
 +
</p>
-
<br>
+
<b>Design your primers with PHUSER <a href="http://igem.grafiki.org/" CLASS=leftbar target="blank">here</a></b><br><br>
 +
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<td width="182px" height="100%" valign="top" bgcolor=DDDDDD class="greybox">
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  <font face="arial" size="2">
 +
 +
 +
<!-- INSERT GREY BOX TEXT HERE! (formatting: <b>bold</> <i>italic> <h4>header</h4>) -->
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 +
 +
<p>
 +
<strong>Achievements</strong><br>
<br>
<br>
 +
<strong>Redox sensing device</strong><br>
 +
Two novel genes have been designed and synthesized each comprised of 5 genetic elements. Together they function as a device termed the Redoxilator that can sense the internal redox state of a yeast cell, and output a reporter signal. Extensive mathematical modelling was performed to simulate how the construct would operate <i>in vivo</i>.<br>
<br>
<br>
 +
<strong>Biobricks</strong><br>
 +
DNA of several new biobricks have been designed and submitted including a yeast optimized GFP reporter protein, a protein degradation sequence and a fast degradable yeast GFP. (Bronze medal)<br>
<br>
<br>
-
+
We have demonstrated that our USER fusion biobrick works as expected and documented it (silver medal)<br>
-
+
<br>
-
+
<strong>USER fusion Assembly standard</strong><br>
-
Biobricks can be fused directly into the standard plasmid containing the USER biobrick.
+
A new biobrick assembly standard that allows the rapid construction of multi-part devices have been developed and documented. The assembly standard offers many benefits: All restriction sites are allowed, multiple biobricks can be joined in one step, the result is scar-free making it ideal for protein fusions and more. (Gold medal)<br>
-
+
<br>
-
+
<strong>USER-fusion primer design software</strong><br>
-
+
A novel and very useful software tool have been developed that can automatically design the optimal primers for USER fusion assembly of 2-9 biobricks, taking several parameters into account.
-
USER fusion relies on the simultaneously fusion and cloning of multiple PCR fragments
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<strong>USER fusion of biobricks - how it works</strong>
+
<td width="967px" valign="center" align="center">
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-
</p>
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<font face="arial" size="1">
 +
Comments or questions to the team? Please <a href="mailto:igem@bio.dtu.dk" CLASS=email>Email us</a> -- Comments of questions to webmaster? Please <a href="mailto:lronn@bio.dtu.dk" CLASS=email>Email us</a>
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Latest revision as of 03:13, 22 October 2009

Wiki banner 967px.png

The project

Home The Team The Project Parts submitted Modelling Notebook


The redoxilator

- Genetic design
- Applications and perspectives
- Safety considerations


The USER assembly standard

- USER fusion of biobricks


USER fusion primer design software

- Abstract
- Instructions
- Output format


Experimental results

- Results and discussion
The project

 The USER fusion assembly standard

The USER fusion assembly standard allows rapid construction of multi-part devices, without some of the drawbacks of the restriction-enzyme based standard biobrick assembly method. The full USER assembly standard can be found here: (BBF RFC 39). The main advantages of this assembly method is:

1. Standardized method for assembling several BioBricks or components at a time, in contrast to the one at a time" assembly procedure normally used.
2. Since the method relies PCR based assembly, all restriction sites are allowed in the biobricks.
3. Eight basepair-overhangs allows ligase-free cloning. With the enclosed protocol E. coli can be transformed with a multipart-construct less than 2 hours after your PCR-reaction has completed.
4. The biobricks are joined without leaving a scar which is ideal for fusing protein domain biobricks.
5. Insertions of small sequences between biobricks such as a intracellular localization signal, restriction site or flexible linker is possible with the right primer design.
6. High fidelity is ensured by using PfuTurbo® Cx Hotstart DNA polymerase.
7. By the design of the PCR tails, it can be decided whether the USER cassette should be deleted, copied or moved following insertion.
8. Directionality of inserts are supported.


USER fusion of biobricks - how it works
We have successfully constructed a USER fusion biobrick (BBa_K194003), and demonstrated that it works as expected. The biobrick includes a DNA-sequence needed for USER-fusion, which consists of a restriction site and two nicking sites. The entire process of fusing two biobricks are illustrated in the figure, and the same can be done for multiple fragments at once.

Principle of USER fusion


Procedure (please refer to our Biobrick Assembly Standard BBF RFC 39 for a detailed protocol):

1. The USER fusion biobrick plasmid is digested with the restriction enzyme pacI and the nicking enzyme Nt.BbvCI (a nicking enzyme cuts only one strand as illustrated on the figure). This process will linearize the plasmid, and make single stranded overhangs (sticky ends).
2. PCR amplification is performed on the biobricks intended for the fusion. The primer design is facilitated by our novel USER fusion primer design software made for this iGEM project.
3. USER enzyme mix is added. This will remove the uracil always included in the primers, making sticky end overhangs on all biobricks. Because of the matching sticky ends on all biobricks and linearized plamid, the biobricks will self-assemble in the plasmid.
Two or more biobricks have been joined with all the advantages mentioned above.

Design your primers with PHUSER here

Achievements

Redox sensing device
Two novel genes have been designed and synthesized each comprised of 5 genetic elements. Together they function as a device termed the Redoxilator that can sense the internal redox state of a yeast cell, and output a reporter signal. Extensive mathematical modelling was performed to simulate how the construct would operate in vivo.

Biobricks
DNA of several new biobricks have been designed and submitted including a yeast optimized GFP reporter protein, a protein degradation sequence and a fast degradable yeast GFP. (Bronze medal)

We have demonstrated that our USER fusion biobrick works as expected and documented it (silver medal)

USER fusion Assembly standard
A new biobrick assembly standard that allows the rapid construction of multi-part devices have been developed and documented. The assembly standard offers many benefits: All restriction sites are allowed, multiple biobricks can be joined in one step, the result is scar-free making it ideal for protein fusions and more. (Gold medal)

USER-fusion primer design software
A novel and very useful software tool have been developed that can automatically design the optimal primers for USER fusion assembly of 2-9 biobricks, taking several parameters into account.

Comments or questions to the team? Please -- Comments of questions to webmaster? Please

Retrieved from "http://2009.igem.org/Team:DTU_Denmark/USERprinciple"