Difference between revisions of "Team:Duke/Experiments"

Revision as of 01:36, 19 October 2016

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.

SDS-PAGE Electrophoresis and Visualization

10% Polyacrylamide tris-glycine gels were either run before or concurrently with the kinetic assays. A Bradford was usually done before a protein gel was attempted. After finding the results of the Bradford, protein was either diluted to 20 ng in each well (or in different ratios if a Bradford was not able to be completed). Samples were heated at 95℃ for 5-10 min to denature the proteins and then loaded. Samples were mixed with 10 uL of sample buffer previous to heating. Once the gel was loaded it was run, between 1.5 and 3 hours, until the samples were either all the way down the gel or the samples stopped running down the gel. Gels were visualized by staining with coomassie between one hour to overnight and then sitting in destain until the bands were visible around 12 hours depending upon the length of time in coomassie.

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.

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 <p> 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.   </p>
 <h4> SDS-PAGE Electrophoresis and Visualization </h4>
-<p> 10% Polyaclamide tris-glycine gels were either run before or concurrently with the kinetic assays. A Bradford was usually done before a protein gel was attempted. After finding the results of the Bradford, protein was either diluted to 20 ng in each well (or in different ratios if a Bradford was not able to be completed). Samples were heated at 95℃ for 5-10 min to denature the proteins and then loaded. Samples were mixed with 10 uL of sample buffer previous to heating. Once the gel was loaded it was run, between 1.5 and 3 hours, until the samples were either all the way down the gel or the samples stopped running down the gel. Gels were visualized by staining with coomassie between one hour to overnight and then sitting in destain until the bands were visible around 12 hours depending upon the length of time in coomassie.   </p>
+<p> 10% Polyacrylamide tris-glycine gels were either run before or concurrently with the kinetic assays. A Bradford was usually done before a protein gel was attempted. After finding the results of the Bradford, protein was either diluted to 20 ng in each well (or in different ratios if a Bradford was not able to be completed). Samples were heated at 95℃ for 5-10 min to denature the proteins and then loaded. Samples were mixed with 10 uL of sample buffer previous to heating. Once the gel was loaded it was run, between 1.5 and 3 hours, until the samples were either all the way down the gel or the samples stopped running down the gel. Gels were visualized by staining with coomassie between one hour to overnight and then sitting in destain until the bands were visible around 12 hours depending upon the length of time in coomassie.   </p>
 <h4> Enzyme Kinetics Assays </h4>
 <p> 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.