Difference between revisions of "Part:BBa K2019000:Design"

 
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===Design Notes===
 
===Design Notes===
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We linearized a backbone with the tet promoter and then used gibson assembly to attach two gblocks with the Cas9 sequence.
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We linearized a backbone with the tet promoter and then used gibson assembly to attach two gblocks with the saCas9 sequence codon optimized for E. Coli.  Then, we used to PCR to amplify out saCas9 with overhangs that matched the homology region on the pET28a backbone.  We then performed a 2 piece Gibson assembly of saCas9 with homology to pET28 and the linearized pET28a backbone.  Finally, we PCR'd saCas9 out of the tet backbone with overhangs that attached the biobricks prefix and suffix to the ends of saCas9.  With this piece, we performed a restriction digestion of our saCas9 PCR product into the linearized PSB1C3 backbone.
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+
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===Source===
 
===Source===
  
−
Tet promoter was used from the .  SaCas9 was extracted from the genomic sequence of staphylococcus aureus and then codon optimized for E. Coli.
+
SaCas9 was extracted from the genomic sequence of staphylococcus aureus and then codon optimized for E. Coli.
  
 
===References===
 
===References===
 +
F. Ann Ran, Le Cong, Winston X. Yan, David A. Scott, Jonathan S. Gootenberg, Andrea J. Kriz, Bernd Zetsche, Ophir Shalem, Xuebing Wu, Kira S. Makarova, Eugene V. Koonin, Phillip A. Sharp, Feng Zhang.  In vivo genome editing using Staphylococcus aureus Cas9.  Nature. 2015;520.
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Friedland AE, Baral R, Singhal P, Loveluck K, Shen S, Sanchez M, Marco E, Gotta GM, Maeder ML, Kennedy EM, Kornepati AVR, Sousa A, Collins MA, Jayaram H, Cullen BR, Bumcrot D. Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications. Genome Biol. 2015;16(257).

Latest revision as of 23:36, 12 October 2016


Histidine Tagged WT Staphylococcus Aureus Cas9 Protein


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 627
    Illegal BglII site found at 780
    Illegal BglII site found at 926
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 200
    Illegal AgeI site found at 2756
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

We linearized a backbone with the tet promoter and then used gibson assembly to attach two gblocks with the saCas9 sequence codon optimized for E. Coli. Then, we used to PCR to amplify out saCas9 with overhangs that matched the homology region on the pET28a backbone. We then performed a 2 piece Gibson assembly of saCas9 with homology to pET28 and the linearized pET28a backbone. Finally, we PCR'd saCas9 out of the tet backbone with overhangs that attached the biobricks prefix and suffix to the ends of saCas9. With this piece, we performed a restriction digestion of our saCas9 PCR product into the linearized PSB1C3 backbone.

Source

SaCas9 was extracted from the genomic sequence of staphylococcus aureus and then codon optimized for E. Coli.

References

F. Ann Ran, Le Cong, Winston X. Yan, David A. Scott, Jonathan S. Gootenberg, Andrea J. Kriz, Bernd Zetsche, Ophir Shalem, Xuebing Wu, Kira S. Makarova, Eugene V. Koonin, Phillip A. Sharp, Feng Zhang. In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015;520.

Friedland AE, Baral R, Singhal P, Loveluck K, Shen S, Sanchez M, Marco E, Gotta GM, Maeder ML, Kennedy EM, Kornepati AVR, Sousa A, Collins MA, Jayaram H, Cullen BR, Bumcrot D. Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications. Genome Biol. 2015;16(257).