Difference between revisions of "Part:BBa K360023:Design"
(→‎Design Notes)
 
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1. The 2 '''PstI restriction sites''' were '''removed''' from the coding region of LovTap.
 
1. The 2 '''PstI restriction sites''' were '''removed''' from the coding region of LovTap.
  
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2. We included a punctual mutation to change the '''ILE427 by a PHE427''', as was proposed by the model  [http://2009.igem.org/Team:EPF-Lausanne/Results/Mutations#ILE427_to_PHE  Simulations and results of predicted lovTAP mutations results of the team iGEM09_EPF-Lausanne]. With this mutation LovTAP should react faster and the conformational change should be more stable (the protein stays in the active form for longer, under light induction).
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2. We included a punctual mutation to change the '''ILE427 by a PHE427''', as was proposed by the model  [http://2009.igem.org/Team:EPF-Lausanne/Results/Mutations#ILE427_to_PHE  results of the team iGEM09_EPF-Lausanne]. With this mutation LovTAP should react faster and the conformational change should be more stable (the protein stays in the active form for longer, under light induction).
  
 
The reason of the conformational change is the following:
 
The reason of the conformational change is the following:
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3. The stop codon tga was changed for two taa.
 
3. The stop codon tga was changed for two taa.
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===Source===
 
===Source===

Latest revision as of 07:48, 21 October 2010

trpL promoter


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 23
    Illegal SpeI site found at 31
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 23
    Illegal SpeI site found at 31
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 23
    Illegal SpeI site found at 31
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 23
    Illegal SpeI site found at 31
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

We decided to synthesize a new LovTap part, that in comparison with the Part:BBa_K191006 that is already at the registry, has the following differences:

1. The 2 PstI restriction sites were removed from the coding region of LovTap.

2. We included a punctual mutation to change the ILE427 by a PHE427, as was proposed by the model [http://2009.igem.org/Team:EPF-Lausanne/Results/Mutations#ILE427_to_PHE results of the team iGEM09_EPF-Lausanne]. With this mutation LovTAP should react faster and the conformational change should be more stable (the protein stays in the active form for longer, under light induction).

The reason of the conformational change is the following:

Cys450 side chain is involved in light state in bond formation with the cofactor. Cys450 can assume two conformational states, called here ON and OFF, and corresponding respectively, to the Sg being near or far from FMN.

The isoleucine 427 is quite big. But not enough to push the cystein's side chain significantly toward the cofactor. So we choose to replace this ILE427 by an PHE427, an amino acid which is much bigger and have more or less the same propreties than the ILE.


3. The stop codon tga was changed for two taa.

Source

LovTAP was assembled by Strickland et al.


References

Strickland, D., Moffat, K., & Sosnick, T. (2008). Light-activated DNA binding in a designed allosteric protein. Proceedings of the National Academy of Sciences, 105(31), 10709. National Acad Sciences. Retrieved from http://www.pnas.org/content/105/31/10709.full.