Part:BBa_K2807011
GLB1 TCT 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 is created by substituting the amino acid at position 107 from phenylalanine to leucine (F107->L107), leading to 1.7% of WT ꞵ-galactosidase 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. At the end of the coding sequence of GLB1, there is a coding sequence for Flag tag and hence, the GLB1 protein expression can be easily identified using the Flag tag.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE 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 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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