Difference between revisions of "Part:BBa K1723005:Design"

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===Design Notes===
 
===Design Notes===
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This part was synthesized on the model of the PAM rich URS J23117 promoter (BBa_K1723001). It was obtained by mutating bases on the promoter to obtain different target sites for sgRNAs for promoter regulation using dCas9-ω system. We mutated the sequences as we wanted except for the -35 sequence and -10 sequence and -35 sequence to keep a similar promoter strength [1].
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This part was synthesized on the model of the PAM rich URS J23117 promoter (BBa_K1723001) [1]. The promoter BBa_K1723001 was mutated to change the sequences of the sgRNA-targeted sites therefore allowing the presence of two independently regulated promoters. We avoided mutating -35 sequence and -10 sequence to keep a similar promoter strength [1] but we mutated inside [2] those sequences. We also mutated outside those sequences which was not tried before. the new targeted sequences were generated randomly and blasted on ''E. coli'' genome to avoid unwanted interactions. Using this procedure we believe more similar promoters could be made. In our experiments the promoter is 40bp upstream of the ATG start codon.
  
 
===Source===
 
===Source===
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[1] Bikard, D., Jiang, W., Samai, P., Hochschild, A., Zhang, F., & Marraffini, L. A. (2013). Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic acids research, 41(15), 7429-7437.
 
[1] Bikard, D., Jiang, W., Samai, P., Hochschild, A., Zhang, F., & Marraffini, L. A. (2013). Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic acids research, 41(15), 7429-7437.
  
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[2] Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., & Lim, W. A. (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 152(5), 1173-1183.
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[2] Alec AK Nielsen & Christopher A Voigt (2014). Multi-input CRISPR/Cas circuits that interface host regulatory network. Molecular systems biology, 10(11), 763.
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[3] Alec AK Nielsen & Christopher A Voigt (2014). Multi-input CRISPR/Cas circuits that interface host regulatory network. Molecular systems biology, 10(11), 763.
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Latest revision as of 22:11, 17 September 2015


PAM rich URS J23117Alt promoter


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 277
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 167
    Illegal XhoI site found at 195
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This part was synthesized on the model of the PAM rich URS J23117 promoter (BBa_K1723001) [1]. The promoter BBa_K1723001 was mutated to change the sequences of the sgRNA-targeted sites therefore allowing the presence of two independently regulated promoters. We avoided mutating -35 sequence and -10 sequence to keep a similar promoter strength [1] but we mutated inside [2] those sequences. We also mutated outside those sequences which was not tried before. the new targeted sequences were generated randomly and blasted on E. coli genome to avoid unwanted interactions. Using this procedure we believe more similar promoters could be made. In our experiments the promoter is 40bp upstream of the ATG start codon.

Source

this part was fully synthesized.

References

[1] Bikard, D., Jiang, W., Samai, P., Hochschild, A., Zhang, F., & Marraffini, L. A. (2013). Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic acids research, 41(15), 7429-7437.

[2] Alec AK Nielsen & Christopher A Voigt (2014). Multi-input CRISPR/Cas circuits that interface host regulatory network. Molecular systems biology, 10(11), 763.