Team:Johns Hopkins/Notebook/BCAssay

From 2011.igem.org

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(→9/9/2011 - 9/12/2011)
(→9/13/2011 - 9/18/2011)
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Took aliquots at different time points to determine the cell count and amount of beta carotene produced.  The cell count was determined by measuring the optical density of the samples at 600nm.   
Took aliquots at different time points to determine the cell count and amount of beta carotene produced.  The cell count was determined by measuring the optical density of the samples at 600nm.   
After addition of pyrogallol in methanol, cells were saponified with potassium hydroxide. The samples were incubated at 75 degrees for an hour.  Beta carotene was then extracted by adding hexane to the samples and shaking them vigorously. Aliquots were then taken for spectroscopy and for drying.
After addition of pyrogallol in methanol, cells were saponified with potassium hydroxide. The samples were incubated at 75 degrees for an hour.  Beta carotene was then extracted by adding hexane to the samples and shaking them vigorously. Aliquots were then taken for spectroscopy and for drying.
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Concentration of beta carotene in hexane was determined from absorbance at 449nm on a UV spectrophotometer.   
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Concentration of beta carotene in hexane was determined from absorbance at 449nm on a UV spectrophotometer.
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{| border="1" cellpadding="5" cellspacing="0" style="margin-left:40px;"
 +
|- style="background-color:#EBEBE9;"
 +
| Strain
 +
| OD600
 +
| OD449
 +
|-
 +
| WT
 +
| 0.895 
 +
|  0 
 +
|-
 +
| 39   
 +
|  0.228
 +
|  0 
 +
|-
 +
|  128   
 +
|  0.226 
 +
|    0
 +
|-
 +
|  179     
 +
|  0.105 
 +
|    0 
 +
|-
 +
|  201
 +
|  0.201
 +
|  0.061
 +
|-
 +
|
 +
|  1:4 dil
 +
|  625 ul for extraction
 +
|}
=====9/19/2011=====
=====9/19/2011=====

Revision as of 18:33, 27 September 2011

VitaYeast - Johns Hopkins University, iGEM 2011

Related Links:

Beta Carotene Extraction
9/9/2011 - 9/12/2011

Extracted beta carotene for different time points for analysis. Found out that carotenoids concentration was too low. Troubleshooted and optimized the procedure for extraction. Grew up a new stock of wild type and beta carotene producing yeasts (Strain 39, 128, 179, 201) in liquid YPD.

9/12/2011 7pm Inoculation

Strain OD600 Hexane OD449 Stock added (ul) YPD volume (ml)
WT 0.044 0 70 20
39 0.156 0 285 20
128 0.205 0 285 20
179 0.212 0 285 20
201 0.192 0 285 20
9/13/2011 - 9/18/2011

Took aliquots at different time points to determine the cell count and amount of beta carotene produced. The cell count was determined by measuring the optical density of the samples at 600nm. After addition of pyrogallol in methanol, cells were saponified with potassium hydroxide. The samples were incubated at 75 degrees for an hour. Beta carotene was then extracted by adding hexane to the samples and shaking them vigorously. Aliquots were then taken for spectroscopy and for drying. Concentration of beta carotene in hexane was determined from absorbance at 449nm on a UV spectrophotometer.

Strain OD600 OD449
WT 0.895 0
39 0.228 0
128 0.226 0
179 0.105 0
201 0.201 0.061
1:4 dil 625 ul for extraction
9/19/2011

Created a calibration curve was used to estimate the number of cells by counting the number of cells counted on a hemocytometer was plotting it against their absorbance. Cell counts at different time points are then compared to it. Beta carotene in hexane was dried in a vacuum desiccator and stored for future use.

9/23/2011

Ran HPLC analysis on carotenoid extracts

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