In order to break down lignocellulose into glucose molecules, we have planned to construct three pathways, aiming at: a) lignocellulose pretreatment/lignin degradation, b) cellulose degradation, and c) hemicellulose/xylan degradation. For each pathway to work, multiple enzymes are needed to work collaboratively within each pathway. We used standard cloning methods to construct
…Biobrick which contains …cellulase?
Cell Surface Codisplay
We constructed a whole-cell biocatalyst with the ability to induce synergistic and sequential cellulose and xylan degradation reactions through codisplay of four types of cellulolytic enzymes on the cell surface of the yeast Pichia pastoris. Cell surface display technique fuses foreign protein with the surface of microorganisms and retains its bioactivity. A yeast strain codisplaying endoglucanase II and cellobiohydrolase II showed significantly will have higher hydrolytic activity and catalyst efficiency with amorphous cellulose than one displaying only endoglucanase II. We utilized the cell surface display technique to simultaneously codisplay ….ase, …ase as individual fusion proteins with the C-terminal-half region of α-agglutinin. Codisplay of the three enzymes on the cell surface was confirmed by observation of immunofluorescence-labeled cells with a fluorescence microscope, which will be introduced later.
Ethanol Fermentation
In order to convert glucose into ethanol and thereby produce cellular energy, we have planned to incubate our engineered yeast cells for the fermentation. Ethanol fermentation is classified as anaerobic because yeasts perform this conversion in the absence of oxygen.
Detection
n order to make sure that several of our cell display enzymes are actually displayed on the surface of yeast, we utilized Immunofluorescence technique to detect protein location and relative abundance. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific protein targets within a cell, and therefore allows detection and visualization of the distribution of our target proteins.We purchased antibodies ( ) for …., which only recognized ….
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