Difference between revisions of "Part:BBa K3219002:Design"

 
 
Line 7: Line 7:
  
 
===Design Notes===
 
===Design Notes===
−
The stop condon of the GFP was silently mutated to allow continuous expression of the dCas9.  
+
The stop codon of GFP is silently mutated to allow continuous expression of the GFP-dCas9 complex.
 +
The shuttle vector is from team 2016 iGEM team Nanjing_NFLS, part BBa_K1894001. It consists of two origin of replications, f1 ori and pUC ori, which allows it to replicate in both E.coli cells and cyanobacteria cells. It also consists of a t7 promoter, CaMV35S promoter, and Kanamycin resistance gene. The T7 promoter allows expression of the dCas9-GFP-sgRNA in E.coli and the CaMVS promoter allows expression in cyanobacteria.
  
 +
The dCas9, also known as a catalytically dead Cas9 enzyme, is a mutated Cas9 enzyme without endonuclease activity. With the help of a single-guide RNA (sgRNA), it specifically binds to the target sequences and blocks transcript elongation by RNA polymerase. The GFP is added to the C-terminus of the dCas9, connected using a linker. There is also a 7x His-Tag, allowing for protein purification.
 +
 +
The sgRNA consists of a handle, a base-pairing region and a terminator. The base-pairing region is designed to target 25 base pairs of the McyB gene of Microcystis Aeruginosa UTEX2388.
  
  

Latest revision as of 10:54, 12 October 2019


Plasmid for in vivo expression of dCas9-sgRNA targeting mcyB


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 3964
    Illegal NheI site found at 12275
    Illegal NotI site found at 777
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1685
    Illegal XhoI site found at 716
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 7979
    Illegal NgoMIV site found at 8139
    Illegal NgoMIV site found at 11186
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 5721
    Illegal SapI site found at 9059


Design Notes

The stop codon of GFP is silently mutated to allow continuous expression of the GFP-dCas9 complex. The shuttle vector is from team 2016 iGEM team Nanjing_NFLS, part BBa_K1894001. It consists of two origin of replications, f1 ori and pUC ori, which allows it to replicate in both E.coli cells and cyanobacteria cells. It also consists of a t7 promoter, CaMV35S promoter, and Kanamycin resistance gene. The T7 promoter allows expression of the dCas9-GFP-sgRNA in E.coli and the CaMVS promoter allows expression in cyanobacteria.

The dCas9, also known as a catalytically dead Cas9 enzyme, is a mutated Cas9 enzyme without endonuclease activity. With the help of a single-guide RNA (sgRNA), it specifically binds to the target sequences and blocks transcript elongation by RNA polymerase. The GFP is added to the C-terminus of the dCas9, connected using a linker. There is also a 7x His-Tag, allowing for protein purification.

The sgRNA consists of a handle, a base-pairing region and a terminator. The base-pairing region is designed to target 25 base pairs of the McyB gene of Microcystis Aeruginosa UTEX2388.


Source

The shuttle vector comes from iGEM part BBa_K1894001. The dCas9 is from the Streptococcus pyogenes Type II CRISPR/Cas system. The GFP is from Aequoria victoria. The sgRNA base-pairing region of the sgRNA is designed to target 25 base pairs of Microcystis Aeruginosa UTEX2388.

References