Team:DTU Denmark/USERprogram

From 2009.igem.org

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<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, 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>
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<p align="justify"><i>Article submitted October 2009 - publication pending</i></p>
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<!-- <p align="justify"><i>Article submitted October 2009 - publication pending</i></p> -->
<p>Questions or comments? Please <a href="mailto:lronn@bio.dtu.dk" CLASS=email>Email us</a></p><br>
<p>Questions or comments? Please <a href="mailto:lronn@bio.dtu.dk" CLASS=email>Email us</a></p><br>

Revision as of 09:03, 21 October 2009

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The project

Home The Team The Project Parts submitted Modelling Notebook

The redoxilator

 - Genetic design
- Applications and perspectives
- Results
- Safety considerations
The USER assembly standard

 - USER fusion of biobricks
USER fusion primer design software

 - Abstract
- Instructions
- Output format
The project


 The USER fusion primer design software: PHUSER
(Primer Help for USER)

Abstract

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 (TM) of the primer fragments must be pairwise within 2 oC 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 TM of primer pairs is done by sampling the various allowed lengths of the primers (18-24 bases), thus changing the CG ration and affecting TM, 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.


Questions or comments? Please


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.

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