Team:Johns Hopkins/Notebook/BCAssay

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Latest revision as of 23:15, 17 October 2011

VitaYeast - Johns Hopkins University, iGEM 2011

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.

Time point 1: 9/13/2011 2pm

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

Time point 2: 9/14/2011 3pm

Strain	OD600	OD449
WT	1.137	0
39	1.158	0.003
128	1.256	0.493
179	0.493	0.005
201	1.248	0.008
	1:10 dil	900 ul for extraction

Time point 3: 9/15/2011 12.30pm

Strain	OD600	OD449
WT	0.364	0
39	0.094	0.011
128	0.106	0
179	0.422	0.02
201	0.107	0.01
	1:50 dil	980 ul for extraction

Time point 4: 9/16/2011 3pm

Strain	OD600	OD449
WT	0.176	0
39	0.228	0.0031
128	0.248	0.002
179	0.197	0.0042
201	0.257	0.038
	1:100 dil	990 ul for extraction

Time point 5: 9/17/2011 11.30pm

Strain	OD600	OD449
WT	0.202	0.004
39	0.318	0.0032
128	0.253	0.017
179	0.227	0.048
201	0.277	0.102
	1:100 dil	990 ul for extraction

Time point 6: 9/17/2011 ~2.30pm

Strain	OD600	OD449
WT	0.150	0.002
39	0.024	0.028
128	0.250	0.006
179	0.269	0.028
201	0.292	0.055
	1:100 dil	740 ul for extraction

Time point 7: 9/18/2011 ~2pm

Strain	OD600	OD449
WT	0.104	0.001
39	0.210	0.022
128	0.233	0.01
179	0.213	0.02
201	0.209	0.041
	1:100 dil	740 ul for extraction

9/19/2011

Created a calibration curve that was used to estimate the number of cells. Counted the number of cells on a hemocytometer and plotted it against the absorbance. Cell counts at different time points were 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 overnight.

9/24/2011

Obtained results from HPLC. Plotted a standard curve using the beta carotene standards, and compared samples to it.

Calibration curve	Beta carotene standard, 70ug/ml	Beta carotene standard, 35ug/ml	Beta carotene standard, 17.5ug/ml
Beta carotene standard, 8.75ug/ml

strain 39, time point 1	strain 39, time point 2	strain 39, time point 3	strain 39, time point 5
strain 39, time point 6

strain 179, time point 1

strain 179, time point 2

strain 179, time point 3

strain 179, time point 5

strain 201, time point 1	strain 201, time point 2	strain 201, time point 3	strain 201, time point 4
strain 201, time point 5	strain 201, time point 6

9/25/2011

First take on extracting beta carotene from bread.

9/26/2011

Repeated extraction for both bread baked with wild type and beta carotene producing yeasts. Around 10g of bread was homogenized with water, 20ml of hexane, 14ml of acetone, and 12ml of ethanol added to solubilize. 8ml of KOH in methanol added to saponify. Another 30ml of hexane and 7ml of toluene added after an hour. 10ml aliquot taken from each sample for HPLC analysis on 9/30.

10/17/2011

Ashan: Inoculated tubes with Omar's mCherry transformant yeast colonies and a 4 tubes with a control group of untransformed yeast colonies of the same strains (201, 039, 179, 128). Left overnight in 30 degree shaker.

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+		<td>
+			<a href="https://2011.igem.org/Team:Johns_Hopkins/Notebook/BCAssay"><img src="https://static.igem.org/mediawiki/2011/6/66/Menu2vit_03.jpg" width="177" height="35" alt=""></a></td>
+		<td>
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+		<td>
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+		<td>
+			<a href="https://2011.igem.org/Team:Johns_Hopkins/Notebook/Baking"><img src="https://static.igem.org/mediawiki/2011/7/72/Menu2vitblack_07.jpg" width="83" height="35" alt=""></a></td>
+	</tr>
+</table></center>
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-Time point 6: 9/17/2011
+Time point 6: 9/17/2011 ~2.30pm
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-Time point 7: 9/18/2011
+Time point 7: 9/18/2011 ~2pm
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 Obtained results from HPLC.  Plotted a standard curve using the beta carotene standards, and compared samples to it.
-[[File:Hplc calibration.png|200px|thumb|Calibration curve]]
+<gallery>
-[[File:4-1.png|200px|thumb|strain 179, time point 1]]
+File:Hplc calibration.png|Calibration curve
-[[File:4-2.png|200px|thumb|strain 179, time point 2]]
+File:Standard 2.png|Beta carotene standard, 70ug/ml
-[[File:4-3.png|200px|thumb|strain 179, time point 3]]
+File:Standard 3.png|Beta carotene standard, 35ug/ml
-[[File:4-5.png|200px|thumb|strain 179, time point 5]]
+File:Standard 4.png|Beta carotene standard, 17.5ug/ml
+File:Standard 5.png|Beta carotene standard, 8.75ug/ml
+</gallery>
+<gallery>
+File:2-1.png|strain 39, time point 1
+File:2-2.png|strain 39, time point 2
+File:2-3.png|strain 39, time point 3
+File:2-5.png|strain 39, time point 5
+File:2-6.png|strain 39, time point 6
+</gallery>
+<gallery>
+File:4-1.png|strain 179, time point 1
+File:4-2.png|strain 179, time point 2
+File:4-3.png|strain 179, time point 3
+File:4-5.png|strain 179, time point 5
+</gallery>
+<gallery>
+File:5-1.png|strain 201, time point 1
+File:5-2.png|strain 201, time point 2
+File:5-3.png|strain 201, time point 3
+File:5-4.png|strain 201, time point 4
+File:5-5.png|strain 201, time point 5
+File:5-6.png|strain 201, time point 6
+</gallery>
 =====9/25/2011=====
@@ Line 308: / Line 353: @@
 Repeated extraction for both bread baked with wild type and beta carotene producing yeasts.
 Around 10g of bread was homogenized with water, 20ml of hexane, 14ml of acetone, and 12ml of ethanol added to solubilize.  8ml of KOH in methanol added to saponify.  Another 30ml of hexane and 7ml of toluene added after an hour.  10ml aliquot taken from each sample for HPLC analysis on 9/30.
+=====10/17/2011=====
+Ashan: Inoculated tubes with Omar's mCherry transformant yeast colonies and a 4 tubes with a control group of untransformed yeast colonies of the same strains (201, 039, 179, 128).  Left overnight in 30 degree shaker.
 <html>
 </div>