Full Technical Reports
An improved reporter system based on a novel β-glucuronidase (GUS) from Staphylococcus sp.
PhD thesis by Tuan Anh Nguyen.
- Chapter 1: General Introduction. (228.9 K)
- Chapter 2: Construction of the synthetic gusASsp gene and its variants. (199.3 K)
- Chapter 3: Purification of β-glucuronidases and preparation of their antibodies. (224.2 K)
- Chapter 4: Expression and secretion studies of GUSEco and GUSSsp in yeast. (113.9 K)
- Chapter 5: Expression and secretion studies of GUSEco and GUSSsp in plants. (142.1 K)
- Chapter 6 & 7: Investigation of some applications based on GUSSsp and Conclusion and prospects. (217.7 K)
The GUS reporter system, based on the enzyme beta-glucuronidase (GUS) from E. coli, has been the most widely used tool in plant molecular biology in the last decade. This thesis reports various studies on a different GUS with superior biochemical characteristics, isolated from a soil Staphylococcus sp. bacteria.
Because of the high AT content of the native coding gene, a codon-optimized version of it was constructed and tested in E. coli, yeast and plants. The protein was expressed in E. coliand purified to high homogeneity for biochemical studies and antibody production.
In yeast, both E. coli GUS and Staphylococcus GUS, when provided with the invertase signal peptide, were efficiently secreted, and mostly localized in the periplasmic space. This is the first report of efficient secretion of E. coli GUS in yeast. All Staphylococcus GUS variants (wildtype, N118Q, and C499A mutants) were also secreted with high efficiency.
In plants, only Staphylococcus GUS was secreted when provided with either the GRP or extensin signal peptide. E. coli GUS was not secreted, consistent with earlier reports. The use of an intron-containng version of the synthetic Staphylococcus gusA as an improved reporter in plants was validated. Staphylococcus GUS was shown to offer faster and more sensitive histochemical detection. Its activity was also better detected after tissue fixation, allowing more precise histochemical localization of the enzyme. With secreted Staphylococcus GUS, non-destructive GUS assays were possible with tissues such as calli and roots. Tissue specific manipulation system using secreted Staphylococcus GUS was also demonstrated, with the example of the secreted Staphylococcus GUS driven by a pollen-specific promoter ntp303.