Difference between revisions of "Team:NUS Singapore-Sci/Reporter GLB"
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Over hundreds of single nucleotide mutations have been implicated in this disease. One of the challenges in building a disease model lies in manipulating GLB1 without affecting normal cellular function since it is an endogenous housekeeping gene. The GLB1 deficient fibroblasts may not be easily available for experimental work. Moreover, while the <i>E. coli</i> β-galactosidase gene <i>LacZ</i> is widely used as a reporter in mammalian systems, this bacterial ortholog cannot be reliably used to create accurate representations of human GLB diseases caused by mutations in the gene. Therefore, we want to investigate if overexpression of exogenous GLB1 is possible in mammalian cell lines. We hope to use such an overexpression system as a disease model to test the usefulness of our RESCUE editor, Cas13b-APOBEC to restore the functions of GLB. Furthermore, we aimed to generate a C to T point mutation in GLB1 that causes a loss of enzymatic function without affecting its translation and degradation. | Over hundreds of single nucleotide mutations have been implicated in this disease. One of the challenges in building a disease model lies in manipulating GLB1 without affecting normal cellular function since it is an endogenous housekeeping gene. The GLB1 deficient fibroblasts may not be easily available for experimental work. Moreover, while the <i>E. coli</i> β-galactosidase gene <i>LacZ</i> is widely used as a reporter in mammalian systems, this bacterial ortholog cannot be reliably used to create accurate representations of human GLB diseases caused by mutations in the gene. Therefore, we want to investigate if overexpression of exogenous GLB1 is possible in mammalian cell lines. We hope to use such an overexpression system as a disease model to test the usefulness of our RESCUE editor, Cas13b-APOBEC to restore the functions of GLB. Furthermore, we aimed to generate a C to T point mutation in GLB1 that causes a loss of enzymatic function without affecting its translation and degradation. | ||
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| + | 1. Construction of EGFP-GLB1 reporter plasmid | ||
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| + | We amplified the flag epitope GLB1 from Genscript GLB1 cDNA ORF clone (NM_000404.3) using PCR, then cloned it into pSB1C3 (<a href="">BBa_K2807014</a>) and a mammalian expression vector, pEGFP-C1. Subsequently, we carried out point mutations to the wild-type GLB gene to generate GLB1 TCT mutant (<a href="">BBa_K2807016</a>) and GLB1 CCG mutant (<a href="">BBa_K2807015</a>) which are point mutations that are reported to reduce GLB1 enzymatic activity by more than 95% in patients suffered from GM1 gangliosidosis. To allow normalization of transfection and expression efficiency of GLB1 in cells, we retained EGFP in C1 vector. To eliminate the possibility of the fusion protein to affect enzymatic activity, we separated EGFP and GLB1 by a stop codon and frameshift linker sequence. We also included a Kozak sequence in front of GLB1 too allow effective ribosome binding to translate GLB1. | ||
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| + | 2. Full-length precursor GLB1 and its mutant can be overexpressed in mammalian cells | ||
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| + | To verify the expression of GLB1 WT and mutants, we expressed both plasmids in HEK293T cells and then tested for the protein expression levels by Western blot. Cell lysates were separated on an SDS-PAGE gel, and transferred onto a nitrocellulose membrane for blotting. The plasmids with GLB1 had a Flag epitope tag as well as an EGFP tag preceding the GLB1 coding sequence. We probed the membrane with anti-EGFP, anti-Flag and anti-Actin antibodies. From the Western blot in Figure 1, we can see that GLB1 protein is expressed as a visible band in both GLB WT and the GLB mutants at 84kDa, the expected size of GLB1 precursor, with the non-transfected lane as the negative control. In addition, a 27kDa band corresponding to EGFP was seen for both GLB WT and the GLB mutants, but not the non-transfected control, confirming our transfection efficiency. Therefore, we can conclude that our WT and mutant EGFP-GLB1 construct is able to express both EGFP and precursor GLB1 at the correct length. Moreover, we confirmed that the reported single nucleotide mutation in GLB1 does not change gene translation level or degradation rate. As such, any change in apparent enzymatic activity of the mutant GLB1 is likely to due to a change in the catalytic efficiency of the enzyme but not the amount of enzyme. | ||
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| + | <caption style="font-size:13px;"><i><strong>Figure 1. Western Blot analysis of cell lysates from transfected HEK293T cells expressing GLB1, GLB1 CCG mutant and GLB1 TCT mutant.</strong> Cell lysates were run on SDS-PAGE and probed using antibodies against EGFP, Flag epitope tag and Actin. Anti-actin antibodies were used to probe for the housekeeping gene actin in order to ensure equal protein loading across samples. | ||
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Revision as of 20:51, 15 October 2018
Reporter System
GLB
GLB1 encodes a human lysosomal acid β-galactosidase (GLB), an enzyme that is responsible for the cleavage of terminal β-linked galactose residues from glycoproteins, sphingolipids, keratan sulfate, and other glycoconjugates. The loss of GLB in physiological systems will result in autosomal recessive lysosomal storage diseases such as GM1 gangliosidosis and Morquio B disease (Suzuki et al., 2001).
Over hundreds of single nucleotide mutations have been implicated in this disease. One of the challenges in building a disease model lies in manipulating GLB1 without affecting normal cellular function since it is an endogenous housekeeping gene. The GLB1 deficient fibroblasts may not be easily available for experimental work. Moreover, while the E. coli β-galactosidase gene LacZ is widely used as a reporter in mammalian systems, this bacterial ortholog cannot be reliably used to create accurate representations of human GLB diseases caused by mutations in the gene. Therefore, we want to investigate if overexpression of exogenous GLB1 is possible in mammalian cell lines. We hope to use such an overexpression system as a disease model to test the usefulness of our RESCUE editor, Cas13b-APOBEC to restore the functions of GLB. Furthermore, we aimed to generate a C to T point mutation in GLB1 that causes a loss of enzymatic function without affecting its translation and degradation.
Over hundreds of single nucleotide mutations have been implicated in this disease. One of the challenges in building a disease model lies in manipulating GLB1 without affecting normal cellular function since it is an endogenous housekeeping gene. The GLB1 deficient fibroblasts may not be easily available for experimental work. Moreover, while the E. coli β-galactosidase gene LacZ is widely used as a reporter in mammalian systems, this bacterial ortholog cannot be reliably used to create accurate representations of human GLB diseases caused by mutations in the gene. Therefore, we want to investigate if overexpression of exogenous GLB1 is possible in mammalian cell lines. We hope to use such an overexpression system as a disease model to test the usefulness of our RESCUE editor, Cas13b-APOBEC to restore the functions of GLB. Furthermore, we aimed to generate a C to T point mutation in GLB1 that causes a loss of enzymatic function without affecting its translation and degradation.
1. Construction of EGFP-GLB1 reporter plasmid
We amplified the flag epitope GLB1 from Genscript GLB1 cDNA ORF clone (NM_000404.3) using PCR, then cloned it into pSB1C3 (BBa_K2807014) and a mammalian expression vector, pEGFP-C1. Subsequently, we carried out point mutations to the wild-type GLB gene to generate GLB1 TCT mutant (BBa_K2807016) and GLB1 CCG mutant (BBa_K2807015) which are point mutations that are reported to reduce GLB1 enzymatic activity by more than 95% in patients suffered from GM1 gangliosidosis. To allow normalization of transfection and expression efficiency of GLB1 in cells, we retained EGFP in C1 vector. To eliminate the possibility of the fusion protein to affect enzymatic activity, we separated EGFP and GLB1 by a stop codon and frameshift linker sequence. We also included a Kozak sequence in front of GLB1 too allow effective ribosome binding to translate GLB1.
2. Full-length precursor GLB1 and its mutant can be overexpressed in mammalian cells
To verify the expression of GLB1 WT and mutants, we expressed both plasmids in HEK293T cells and then tested for the protein expression levels by Western blot. Cell lysates were separated on an SDS-PAGE gel, and transferred onto a nitrocellulose membrane for blotting. The plasmids with GLB1 had a Flag epitope tag as well as an EGFP tag preceding the GLB1 coding sequence. We probed the membrane with anti-EGFP, anti-Flag and anti-Actin antibodies. From the Western blot in Figure 1, we can see that GLB1 protein is expressed as a visible band in both GLB WT and the GLB mutants at 84kDa, the expected size of GLB1 precursor, with the non-transfected lane as the negative control. In addition, a 27kDa band corresponding to EGFP was seen for both GLB WT and the GLB mutants, but not the non-transfected control, confirming our transfection efficiency. Therefore, we can conclude that our WT and mutant EGFP-GLB1 construct is able to express both EGFP and precursor GLB1 at the correct length. Moreover, we confirmed that the reported single nucleotide mutation in GLB1 does not change gene translation level or degradation rate. As such, any change in apparent enzymatic activity of the mutant GLB1 is likely to due to a change in the catalytic efficiency of the enzyme but not the amount of enzyme.