Difference between revisions of "Part:BBa K2807010"
Line 5: Line 5:
 
GLB1 encodes a human lysosomal acid beta galactosidase, an enzyme that is responsible for the cleavage of terminal β-linked galactose residues from glycoproteins, sphingolipids, keratan sulfate, and other glycoconjugates. This part was used in our composite reporter part for indicating base editing as single base mutations in GLB1 have been linked to causing disease conditions.  
 
GLB1 encodes a human lysosomal acid beta galactosidase, an enzyme that is responsible for the cleavage of terminal β-linked galactose residues from glycoproteins, sphingolipids, keratan sulfate, and other glycoconjugates. This part was used in our composite reporter part for indicating base editing as single base mutations in GLB1 have been linked to causing disease conditions.  
  
−
As such, GLB1 was mutated using PCR mutagenesis such that it makes the protein non-functional. The point mutation in this CCG mutant is to change the amino acid from I389 to P289. This (I-> P) mutation results in a 99.5% reduction in GLB gene expression. Subsequently, our base editing system can be employed to reverse the mutation made. A successful base repair by Cas13b-APOBEC fusion protein would allow the conversion of a mutant GLB1 to a functional GLB1, thus leading to restoration of enzymatic function.   
+
As such, GLB1 was mutated using PCR mutagenesis such that it makes the protein non-functional. The point mutation in this CCG mutant is to change the amino acid from I389 to P389. This (I-> P) mutation results in a 99.5% reduction in GLB gene expression. Subsequently, our base editing system can be employed to reverse the mutation made. A successful base repair by Cas13b-APOBEC fusion protein would allow the conversion of a mutant GLB1 to a functional GLB1, thus leading to restoration of enzymatic function.   
  
  

Revision as of 05:47, 16 October 2018


GLB1 CCG mutant

GLB1 encodes a human lysosomal acid beta galactosidase, an enzyme that is responsible for the cleavage of terminal β-linked galactose residues from glycoproteins, sphingolipids, keratan sulfate, and other glycoconjugates. This part was used in our composite reporter part for indicating base editing as single base mutations in GLB1 have been linked to causing disease conditions.

As such, GLB1 was mutated using PCR mutagenesis such that it makes the protein non-functional. The point mutation in this CCG mutant is to change the amino acid from I389 to P389. This (I-> P) mutation results in a 99.5% reduction in GLB gene expression. Subsequently, our base editing system can be employed to reverse the mutation made. A successful base repair by Cas13b-APOBEC fusion protein would allow the conversion of a mutant GLB1 to a functional GLB1, thus leading to restoration of enzymatic function.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 10
    Illegal BamHI site found at 41
    Illegal BamHI site found at 1398
    Illegal BamHI site found at 1734
    Illegal XhoI site found at 14
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]