Difference between revisions of "Part:BBa K568001:Design"

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===Design Notes===
 
===Design Notes===
 
We used YcgF/E as blue light sensor and Cph8 as red light sensor. We expect that this assembly will be the one with the lowest amount of unspecific gene expression which could occur due to overlapping of the absorption spectrums of the sensory domains.
 
We used YcgF/E as blue light sensor and Cph8 as red light sensor. We expect that this assembly will be the one with the lowest amount of unspecific gene expression which could occur due to overlapping of the absorption spectrums of the sensory domains.
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+
The red light sensor is active in the ground state, so the signalling needs to be shut down. This can be achieved by irradiation with light of 650 nm.
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+
The signal transduction pathway of the red light sensor is supposedly also responsible for sensing of aspartate, a substance which is present in LB as well as other common media for E. coli. Hence a strain with EnvZ knockout is desireable. For this the strain CP919 (V1012) can be used.
  
 
===Source===
 
===Source===
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Designed from existing Biobricks.
 
Designed from existing Biobricks.
  
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High Fidelity PCR was performed on K322127 (Red Light Sensor Part with lacZ promoter) to amplify the region withouth RBS and LacZ. The PCR product contains pcyA, heme oxygenase, as well as cph8 and ompC promoter (correspond to parts K322123, K322124 and R0082, which form BBa_K568000). This PCR product was assembled with a synthesized construct containing the small parts of the AND-Gate (BBa_K568006). The final step was ligating this intermediate part BBa_K568005 with T7ptag (BBa_K228000).
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High Fidelity PCR was performed on <html><a href="https://parts.igem.org/Part:BBa_K322127">BBa_K322127</a></html> (Red Light Sensor Part with lacZ promoter) to amplify the region withouth RBS and LacZ. The PCR product contains pcyA, heme oxygenase, as well as cph8 and ompC promoter (correspond to parts K322123, K322124 and R0082, which form <html><a href="https://parts.igem.org/Part:BBa_K568000">BBa_K568000</a></html>). This PCR product was assembled with a synthesized construct containing the small parts of the AND-Gate (<html><a href="https://parts.igem.org/Part:BBa_K568006">BBa_K568006</a></html>). The final step was ligating this intermediate part <html><a href="https://parts.igem.org/Part:BBa_K568005">BBa_K568005</a></html> with T7ptag (<html><a href="https://parts.igem.org/Part:BBa_K228000">BBa_K228000</a></html>).
  
 
===References===
 
===References===
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J Christopher Anderson, Christopher A Voigt and Adam P Arkin. Environmental signal integration by a modular and gate. Mol Syst Biol, 3, 08 2007.[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1964800/]]

Latest revision as of 18:38, 21 September 2011

Optogenetical AND-Gate red/blue light


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 644
    Illegal XhoI site found at 1786
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 971
    Illegal AgeI site found at 3817
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 3820


Design Notes

We used YcgF/E as blue light sensor and Cph8 as red light sensor. We expect that this assembly will be the one with the lowest amount of unspecific gene expression which could occur due to overlapping of the absorption spectrums of the sensory domains. The red light sensor is active in the ground state, so the signalling needs to be shut down. This can be achieved by irradiation with light of 650 nm. The signal transduction pathway of the red light sensor is supposedly also responsible for sensing of aspartate, a substance which is present in LB as well as other common media for E. coli. Hence a strain with EnvZ knockout is desireable. For this the strain CP919 (V1012) can be used.

Source

Designed from existing Biobricks.

High Fidelity PCR was performed on BBa_K322127 (Red Light Sensor Part with lacZ promoter) to amplify the region withouth RBS and LacZ. The PCR product contains pcyA, heme oxygenase, as well as cph8 and ompC promoter (correspond to parts K322123, K322124 and R0082, which form BBa_K568000). This PCR product was assembled with a synthesized construct containing the small parts of the AND-Gate (BBa_K568006). The final step was ligating this intermediate part BBa_K568005 with T7ptag (BBa_K228000).

References

J Christopher Anderson, Christopher A Voigt and Adam P Arkin. Environmental signal integration by a modular and gate. Mol Syst Biol, 3, 08 2007.http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1964800/