Difference between revisions of "Part:BBa K2361000:Design"
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===Design Notes===
 
===Design Notes===
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This protein was selected because it lacked its endonuclease activity. If the normal viriant was used, it would cut up our reporter plasmid. This was the initial starting protein as the modifications were made later to target specific sequences related to the Groningen 2017 project and to meet all the biobrick characteristics.
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This is Cas9 with two amino acid substitutions (D10A & H840A) that disrupt its two nuclease domains. If the normal variant was used, it would cut up our reporter plasmid. This was the initial starting protein as the modifications were made later to target specific sequences related to the Groningen 2017 project and to meet all the biobrick characteristics.
  
  

Revision as of 13:56, 12 October 2017


spdCas9


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1100
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 3379
    Illegal XhoI site found at 4115
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This is Cas9 with two amino acid substitutions (D10A & H840A) that disrupt its two nuclease domains. If the normal variant was used, it would cut up our reporter plasmid. This was the initial starting protein as the modifications were made later to target specific sequences related to the Groningen 2017 project and to meet all the biobrick characteristics.


Source

This part originates from the Addgene plasmid pJWV102-PL-dCas9. PCR was used to amplify the protein coding sequence to integrate into a biobrick format.

References

High-throughput CRISPRi phenotyping identifies new essential genes in Streptococcus pneumoniae. Liu X, Gallay C, Kjos M, Domenech A, Slager J, van Kessel SP, Knoops K, Sorg RA, Zhang JR, Veening JW. Mol Syst Biol. 2017 May 10;13(5):931. doi: 10.15252/msb.20167449.