Team:Duke/Experiments

Our project consisted of two main sections, the cloning and the kinetic assays.

Cloning

Protocols

Experiments

Kinetic Assays

Protocols

Preparation of Metabolic Cell Line

To begin the characterization of the enzymes, each recombinant plasmid was transformed into DLF-00286 cells (optimized for metabolic expression). This was accomplished using the transformation protocol used during cloning.

Phosphate Limited Expression

Recombinant genes for the taxol pathway enzymes were expressed using limited-phosphate media to active the phosphate induced promoter. In order to build up biomass for enzymatic analysis, growth of the cells in SM10++ media—a high density (10-15 grams of biomass per liter) growth media—was used. Starter cultures of cells expressing each enzyme were grown overnight at 37℃ and 220 rpm shaking and then transferred into 50 ml of SM10++ in a flat cell culture flasks, which were incubated until sufficient growth. Sufficient growth for harvest was set at an optical density (OD 600) of between 7 and 10. After centrifugation, the cells were then washed with FGM-No Phosphate media prior to resuspension. This was to ensure the removal of residual phosphate remained in the solution. Expression for our genes was inhibited by the presence of phosphate from the growth media. After final resuspension in FGM-No Phosphate, cultures were allowed to express at 37℃ and 220 rpm between 16 and 28 hours. Experimentally, the incubation time was positively correlated with greater protein expression.

Sonication

Once cells had been grown and the enzyme expressed, cells were stored at -80℃ until they were to be used in SDS-PAGE and Kinetic Assays. To begin the sonication process, cells were unfrozen and then mixed with 2-5 mL of Lysate buffer, a mixture of phosphate buffer and protease inhibitors. EDTA was not added to our lysate buffer because our enzymes would react with the EDTA. Once samples were prepared, they were sonicated in a sonicator. Samples were sonicated at max amplitude of the machine using a pulse system with an on cycle of 10 s and and off cycle of 30 s. Total sonication time was between 3 and 5 min. After sonication, the samples were spun at 10,000 rpm for 20 min at 4℃. This was to remove the cell debris from the sample. The supernatant was then placed into a 30 kDa filter and filtered. Each spin was for 2-5 min at max speed permitted by the filter. When half of the sample had been filtered, more lysate buffer was added and mixed to the sample. The samples were washed three times in this manner. Once the samples had been filtered, they were kept on ice until an SDS-PAGE and/or kinetic assays could be attempted.

SDS-PAGE Electrophoresis and Visualization

Electrophoresis using a 10% Polyacrylamide tris-glycine gels (Thermo Fisher) was run to identify the presence of recombinant proteins in the lysates prepared from expression induced E. coli cultures. Prior to loading the gel, a Bradford Protein Quantification Assay was used to quantify the total protein in a sample by comparing it to the bovine serum albumin (BSA) standards. Using the results of the Bradford assay, 20 ng of total protein diluted to 10 μl total volume was mixed with 10 μl 2x Laemmli Blue SDS Sample Buffer in order to denature the proteins. Samples were heated at 95℃ for 3-5 min to further denature proteins and then allowed to cool on ice. The Fisher gel box was assembled with 10% Polyacrylamide tris-glycine gel and filled with 1x Tris Glycine Running Buffer pH 8.0. In each well, 18 μl of each sample mix was loaded along with 10 μl of NEB 10-250 kDa Protein Ladder. The gel was allowed to run for 1 hour and 45 minutes at 180V. Gels were visualized by overnight staining in Coomassie Brilliant Blue. The gel was incubated in destain solution until the appearance of visible bands and the disappearance of background blue color. Bands on the gel were compared to the protein ladder.

Enzyme Kinetics Assays

Assays were attempted for enzymes. Samples were taken at regular intervals - some experiments lasted for several minutes while others lasted for hours. Depending on the enzyme, the ingredients to each assay changed.