Team:DTU Denmark/USERprogram

When designing constructs with more than two biobricks using USER fusion, it is essential to avoid identical fusion tails to ensure correct order of the biobricks. Furthermore, the DNA denaturation temperature (T_M) of the primer fragments must be pairwise within 2 ^oC degrees of each other, for successful PCR amplication of the biobricks. Selection of optimal fusion tails is achieved by employing a simple, but powerful sorting algorithm utilizing the fact that the relative penalty for increasing the length of, and shifting the center of fusion regions, is the same, i.e. one base added/removed from final primers. Adjusting the T_M of primer pairs is done by sampling the various allowed lengths of the primers (18-24 bases), thus changing the CG ration and affecting T_M until an acceptable solution is achieved. The suggested primers are presented in a clear and intuitive fashion, diplaying both list view and a graphical overview of fusion regions and related primers. PHUSER is tested to handle primer design for constructs with between 2 and 9 biobricks at the time, but in theory, if enough unique fusion tails exist, many more biobricks can be fused in the same reaction.

Questions or comments? Please Email us

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 Email us -- Comments of questions to webmaster? Please Email us

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-   <font color="#990000" face="arial" size="3">
+<p>
+ <font color="#990000" face="arial" size="3">
     <br>
-    The redoxilator<br><br>
+    The redoxilator<br>
     </font>
+</p>
     <font color="" face="arial" size="2">
      <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>
-    <a href="https://2009.igem.org/Team:DTU_Denmark/results" CLASS=leftbar>- Results</a><br>
+     <a href="https://2009.igem.org/Team:DTU_Denmark/safety" CLASS=leftbar>- Safety considerations</a><br>
-     <a href="https://2009.igem.org/Team:DTU_Denmark/safety" CLASS=leftbar>- Safety considerations</a><br><br>
     </font>
+<p>
     <font color="#990000" face="arial" size="3">
-     <br>The USER assembly standard<br><br>
+     <br>The USER assembly standard<br>
     </font>
+</p>
     <font face="arial" size="2">
-     <a href="https://2009.igem.org/Team:DTU_Denmark/USERprinciple" CLASS=leftbar>- USER fusion of biobricks</a><br><br>
+     <a href="https://2009.igem.org/Team:DTU_Denmark/USERprinciple" CLASS=leftbar>- USER fusion of biobricks</a><br>
     </font>
+<p>
     <font color="#990000" face="arial" size="3">
-     <br>USER fusion primer design software<br><br>
+     <br>USER fusion primer design software<br>
     </font>
+</p>
     <font face="arial" size="2">
      <a href="https://2009.igem.org/Team:DTU_Denmark/USERprogram" CLASS=leftbar>- Abstract</a><br>
      <a href="https://2009.igem.org/Team:DTU_Denmark/USERprograminstructions" CLASS=leftbar>- Instructions</a><br>
      <a href="https://2009.igem.org/Team:DTU_Denmark/USERprogramoutputformat" CLASS=leftbar>- Output format</a><br>
+   </font>
+<p>
+   <font color="#990000" face="arial" size="3">
+    <br>Experimental results<br>
+   </font>
+</p>
+   <font face="arial" size="2">
+    <a href="https://2009.igem.org/Team:DTU_Denmark/results" CLASS=leftbar>- Results and discussion</a><br>
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-    <b>The project</b><br><br><br>
+    <b>The project</b><br><br>
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 <font size="3"><b>Abstract</b></font><br>
-<p align="justify">When designing constructs with more than two biobricks using USER fusion, it is essential to avoid identical fusion tails to ensure correct order of the biobricks. Furthermore, the DNA denaturation temperature (T<sub>M</sub>) of the primer fragments must be pairwise within 2 <sup>o</sup>C degrees, for PCR amplication of the biobricks. Selection of optimal fusion tails is achieved by employing a simple, but powerful sorting algorithm utilizing the fact that the relative penalty for increasing length of, and shifting center of fusion regions, is the same, i.e. one base added/removed from final primers. Adjusting the T<sub>M</sub> of primer pairs is done by sampling the various allowed lengths of the primers (18-24 bases), thus changing the CG ration and affecting T<sub>M</sub>, until an acceptable solution is achieved. The suggested primers are presented in a clear and intuitive fashion, diplaying both list view and a graphical overview of fusion regions and related primers. PHUSER is tested to handle primer design for constructs with between 2 and 9 biobricks at the time, but in theory, if enough unique fusion tails exists, many more biobricks can be fused in the same reaction.</p><br>
+<p align="justify">When designing constructs with more than two biobricks using USER fusion, it is essential to avoid identical fusion tails to ensure correct order of the biobricks. Furthermore, the DNA denaturation temperature (T<sub>M</sub>) of the primer fragments must be pairwise within 2 <sup>o</sup>C degrees of each other, for successful PCR amplication of the biobricks. Selection of optimal fusion tails is achieved by employing a simple, but powerful sorting algorithm utilizing the fact that the relative penalty for increasing the length of, and shifting the center of fusion regions, is the same, i.e. one base added/removed from final primers. Adjusting the T<sub>M</sub> of primer pairs is done by sampling the various allowed lengths of the primers (18-24 bases), thus changing the CG ration and affecting T<sub>M</sub> until an acceptable solution is achieved. The suggested primers are presented in a clear and intuitive fashion, diplaying both list view and a graphical overview of fusion regions and related primers. PHUSER is tested to handle primer design for constructs with between 2 and 9 biobricks at the time, but in theory, if enough unique fusion tails exist, many more biobricks can be fused in the same reaction.</p><br>
 <!-- <p align="justify"><i>Article submitted October 2009 - publication pending</i></p> -->