Difference between revisions of "Part:BBa M36394:Experience"

Revision as of 03:36, 9 December 2011

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Applications of BBa_M36394

In conducting this project we made use of the Rhamnex 67K Xbrane plasmid. This plasmid contains a rhamnose inducible promoter system, rhaBAD. We inoculated cells containing this plasmid into 3 mL of LB+AMP solution and grew them overnight. Then, we determined the OD600 and added 1000uM rhamnose to give a cell density of 0.01 for the primary assay. After overnight growth in 3mL of LB+AMP with rhamnose added, we spun down 1 mL of the cells at 13,300 G for 5 minutes before resuspending in 100uL of LB+AMP media. We then lysed the cells by adding 60uL of cells to 60uL of 0.1% SDS, 1 mL of Buffer Z, and 50uL of chloroform. We then waited 5 minutes for separation after vortexing for 10 seconds. Next, we added varying cell lysate volumes to Buffer 1 ((150 × 10-3M sodium chloride, 5x10-3M potassium phosphate, pH = 6.87, and 12.5 × 10−6M Congo Red) to give a final volume of 1 mL. Congo red is a dye that binds to amyloid like proteins such as spider silk and allows for absorbance analysis at 541 nm. Negative control solutions of cells from the exact same stock without prior growth in rhamnose media served as a blank for the rhamnose-induced cells. The identical volumes of lysates were added and served as a blank for the respective rhamnose induced sample. (The negative control samples were also spun down and lysed in the same way as the rhamnose induced cells)

Next, we took the ideal concentration of cell lysate from the primary assay, 500uL, and conducted a secondary assay with the exact same parameters as the first except that cells were grown overnight in varying concentrations of rhamnose (100uM, 250uM, 500uM, 750uM, and 1000 uM). Negative control solutions of cells from the same stock that had not been induced with rhamnose were again used as blanks. They were treated identically as the rhamnose induced cells. Results can be seen below.

These images show that from the experiments we conducted, 500uL is the ideal cell lysate volume to add to 500uL of Congo Red in order to maximize absorbance with 1000uM rhamnose.

The Secondary Assay results do not demonstrate such a telling trend, but the data do seem to indicate that still 1000uM of rhamnose gives the highest absorbance after overnight growth. More experiments should produce a nicer trend.

Please let us know if you attempt to isolate the protein or even spin it out!!!

User Reviews

UNIQ79a5c670d72a4b79-partinfo-00000000-QINU UNIQ79a5c670d72a4b79-partinfo-00000001-QINU

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 ===Applications of BBa_M36394===
-Alhough we were unable to isolate spider silk protein produced by the E. coli cells, we did conduct dosage response assays on our system. We made use of Congo Red, a dye that binds to amyloid like proteins such as spider silk, to test for the presence of the protein in cell lysate at 541 nm. Altough we went off the methodology of Slotta et al. for usage of Congo Red, we were unable to isolate the spider silk protein in the limited time for the project. Thus, we conducted a primary assay to determine the ideal volume of cell lysate volume to add to Congo Red buffer before conducting a secondary assay adding this ideal volume of cell lysate to varying concentrations of rhamnose.
+In conducting this project we made use of the Rhamnex 67K Xbrane plasmid. This plasmid contains a rhamnose inducible promoter system, rhaBAD. We inoculated cells containing this plasmid into 3 mL of LB+AMP solution and grew them overnight. Then, we determined the OD600 and added 1000uM rhamnose to give a cell density of 0.01 for the primary assay. After overnight growth in 3mL of LB+AMP with rhamnose added, we spun down 1 mL of the cells at 13,300 G for 5 minutes before resuspending in 100uL of LB+AMP media. We then lysed the cells by adding 60uL of cells to 60uL of 0.1% SDS, 1 mL of Buffer Z, and 50uL of chloroform. We then waited 5 minutes for separation after vortexing for 10 seconds. Next, we added varying cell lysate volumes to Buffer 1 ((150 × 10-3M sodium chloride, 5x10-3M potassium phosphate, pH = 6.87, and 12.5 × 10−6M Congo Red) to give a final volume of 1 mL. Congo red is a dye that binds to amyloid like proteins such as spider silk and allows for absorbance analysis at 541 nm. Negative control solutions of cells from the exact same stock without prior growth in rhamnose media served as a blank for the rhamnose-induced cells. The identical volumes of lysates were added and served as a blank for the respective rhamnose induced sample. (The negative control samples were also spun down and lysed in the same way as the rhamnose induced cells)
+Next, we took the ideal concentration of cell lysate from the primary assay, 500uL, and conducted a secondary assay with the exact same parameters as the first except that cells were grown overnight in varying concentrations of rhamnose (100uM, 250uM, 500uM, 750uM, and 1000 uM). Negative control solutions of cells from the same stock that had not been induced with rhamnose were again used as blanks. They were treated identically as the rhamnose induced cells. Results can be seen below.
 [[Image:primary assay.jpg]]
@@ Line 12: / Line 14: @@
 These images show that from the experiments we conducted, 500uL is the ideal cell lysate volume to add to 500uL of Congo Red in order to maximize absorbance with 1000uM rhamnose.
-The Secondary Assay results do not demonstrate such a telling trend, but the data do seem to indicate that still 1000uM of rhamnose gives the highest absorbance after overnight growth.
+The Secondary Assay results do not demonstrate such a telling trend, but the data do seem to indicate that still 1000uM of rhamnose gives the highest absorbance after overnight growth. More experiments should produce a nicer trend.
 Please let us know if you attempt to isolate the protein or even spin it out!!!