TUDelft/15 October 2008

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=October 15th=
=October 15th=
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<b>This protocol has been discarded due to the sonicator not being capable of reliably lysing >50 samples.</b>
 
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===Cell preparation===
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==Luciferase and protein content measurements==
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*Pellet 750 ml of grown culture when the culture is around 0.9 by centrifuging 5 min at 10000 rpm in a tabletop centrifuge at 4oC.  
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*Resuspend in 1x PBS, keeping the cells on ice.  
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The results of the corrected luciferase/total protein activity is displayed in figure 1. Unfortunately, all the strains grown at 25ºC and 30ºC showed no luciferase activity. We expect lysis of the cells did not work, due to a low amplitude of the sonicator. K115031 is left out here because it has such a high luciferase activity it interferes with the scale. For the full graph click [[TUDelft/081015c.png|here]].
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*Sonicate the cells by using the sonicator at 2 x 15 seconds, with a 15 second interval, at max amplitude. (decreases as we go in our lab)
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===Measurements===
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[[Image:TUDelft221508a.png|thumb|center|400px|Figure 1: Corrected luciferase activity in lum / mg total protein. Errorbars represent N=4. Luciferase measured in duplo.]]
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*Mix 1 ul of 100X luciferase substrate in 100 ul of assay buffer per sample in the luminometer's reagent tube no. 1
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*Put 20 ul of lysed cells in a white 96 wells plate.  
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In figure 2 we displayed the 37ºC fold increase of luminescence / total protein over the 20ºC sample.
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*Put the prime plate in the luminometer (usually on top)
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[[Image:TUDelft221508b.png|thumb|center|400px|Figure 2: Fold increase of luminescence / total protein over the 20ºC sample (normalized for 20ºC).]]
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*Put the tube with assay buffer under reagent needle no. 1, make sure the tip of the needle is in a position to reach all the assay buffer.  
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*Prime the luminometer with 1000 ul assay buffer in the priming plate(make sure the tubing is rinsed before)
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From this experiment we drew the conclusion that construct K115035 is the most probable construct for showing temperature induced switching behavior.
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*Measure luciferase activity by:  
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**Adding 100 ul of assay buffer to a well
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==Growing 25ºC and 30ºC cultures (again)==
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**Wait 2 seconds
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**Integrate luminescence for 10 seconds.
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Due to the failing of lysis of cultures grown at these temperatures, we've started new o/n growth of these cultures. New method of lysis will be the Fastprep machine.  
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**Repeat for every well
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**There is a standard protocol on the computer in which you only have to indicate the wells to be assayed.
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*Purge the tubing (Assay buffer can be stored and frozen for short periods (1 week at most) at -80oC according to the technical manual)
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*Rinse the tubing with 5000 ul of ethanol, and purge it.
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*Rinse the tubing with 5000 ul of H2O, and purge it.
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*The tubing and injector should be clean and empty now.
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Latest revision as of 15:09, 28 October 2008

July
MTWTFSS
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7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30 31
August
MTWTFSS
        1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31

September
MTWTFSS
[http://2008.igem.org/TUDelft/1_September_2008 1] [http://2008.igem.org/TUDelft/2_September_2008 2] [http://2008.igem.org/TUDelft/3_September_2008 3] [http://2008.igem.org/TUDelft/4_September_2008 4] [http://2008.igem.org/TUDelft/5_September_2008 5] [http://2008.igem.org/wiki/index.php?title=TUDelft/6_September_2008&action=edit 6] [http://2008.igem.org/wiki/index.php?title=TUDelft/7_September_2008&action=edit 7]
[http://2008.igem.org/TUDelft/8_September_2008 8] [http://2008.igem.org/TUDelft/9_September_2008 9] [http://2008.igem.org/TUDelft/10_September_2008 10] [http://2008.igem.org/TUDelft/11_September_2008 11] [http://2008.igem.org/TUDelft/12_September_2008 12] [http://2008.igem.org/wiki/index.php?title=TUDelft/13_September_2008&action=edit 13] [http://2008.igem.org/wiki/index.php?title=TUDelft/14_September_2008&action=edit 14]
[http://2008.igem.org/TUDelft/15_September_2008 15] [http://2008.igem.org/TUDelft/16_September_2008 16] [http://2008.igem.org/TUDelft/17_September_2008 17] [http://2008.igem.org/TUDelft/18_September_2008 18] [http://2008.igem.org/TUDelft/19_September_2008 19] [http://2008.igem.org/wiki/index.php?title=TUDelft/20_September_2008&action=edit 20] [http://2008.igem.org/wiki/index.php?title=TUDelft/21_September_2008&action=edit 21]
[http://2008.igem.org/TUDelft/22_September_2008 22] [http://2008.igem.org/TUDelft/23_September_2008 23] [http://2008.igem.org/TUDelft/24_September_2008 24] [http://2008.igem.org/TUDelft/25_September_2008 25] [http://2008.igem.org/wiki/index.php?title=TUDelft/26_September_2008&action=edit 26] [http://2008.igem.org/wiki/index.php?title=TUDelft/27_September_2008&action=edit 27] [http://2008.igem.org/wiki/index.php?title=TUDelft/28_September_2008&action=edit 28]
[http://2008.igem.org/TUDelft/29_September_2008 29] [http://2008.igem.org/TUDelft/30_September_2008 30]
October
MTWTFSS
    [http://2008.igem.org/TUDelft/1_October_2008 1] [http://2008.igem.org/TUDelft/2_October_2008 2] [http://2008.igem.org/TUDelft/3_October_2008 3] [http://2008.igem.org/wiki/index.php?title=TUDelft/4_October_2008&action=edit 4] [http://2008.igem.org/wiki/index.php?title=TUDelft/5_October_2008&action=edit 5]
[http://2008.igem.org/TUDelft/6_October_2008 6] [http://2008.igem.org/TUDelft/7_October_2008 7] [http://2008.igem.org/TUDelft/8_October_2008 8] [http://2008.igem.org/TUDelft/9_October_2008 9] [http://2008.igem.org/TUDelft/10_October_2008 10] [http://2008.igem.org/wiki/index.php?title=TUDelft/11_October_2008&action=edit 11] [http://2008.igem.org/wiki/index.php?title=TUDelft/12_October_2008&action=edit 12]
[http://2008.igem.org/TUDelft/13_October_2008 13] [http://2008.igem.org/TUDelft/14_October_2008 14] [http://2008.igem.org/TUDelft/15_October_2008 15] [http://2008.igem.org/TUDelft/16_October_2008 16] [http://2008.igem.org/TUDelft/17_October_2008 17] [http://2008.igem.org/wiki/index.php?title=TUDelft/18_October_2008&action=edit 18] [http://2008.igem.org/wiki/index.php?title=TUDelft/19_October_2008&action=edit 19]
[http://2008.igem.org/TUDelft/20_October_2008 20] [http://2008.igem.org/TUDelft/21_October_2008 21] [http://2008.igem.org/TUDelft/22_October_2008 22] [http://2008.igem.org/TUDelft/23_October_2008 23] [http://2008.igem.org/TUDelft/24_October_2008 24] [http://2008.igem.org/wiki/index.php?title=TUDelft/25_October_2008&action=edit 25] [http://2008.igem.org/wiki/index.php?title=TUDelft/26_October_2008&action=edit 26]
[http://2008.igem.org/wiki/index.php?title=TUDelft/27_October_2008&action=edit 27] [http://2008.igem.org/wiki/index.php?title=TUDelft/28_October_2008&action=edit 28] [http://2008.igem.org/wiki/index.php?title=TUDelft/29_October_2008&action=edit 29] [http://2008.igem.org/wiki/index.php?title=TUDelft/30_October_2008&action=edit 30] [http://2008.igem.org/wiki/index.php?title=TUDelft/31_October_2008&action=edit 31]

October 15th

Luciferase and protein content measurements

The results of the corrected luciferase/total protein activity is displayed in figure 1. Unfortunately, all the strains grown at 25ºC and 30ºC showed no luciferase activity. We expect lysis of the cells did not work, due to a low amplitude of the sonicator. K115031 is left out here because it has such a high luciferase activity it interferes with the scale. For the full graph click here.

Figure 1: Corrected luciferase activity in lum / mg total protein. Errorbars represent N=4. Luciferase measured in duplo.

In figure 2 we displayed the 37ºC fold increase of luminescence / total protein over the 20ºC sample.

Figure 2: Fold increase of luminescence / total protein over the 20ºC sample (normalized for 20ºC).

From this experiment we drew the conclusion that construct K115035 is the most probable construct for showing temperature induced switching behavior.

Growing 25ºC and 30ºC cultures (again)

Due to the failing of lysis of cultures grown at these temperatures, we've started new o/n growth of these cultures. New method of lysis will be the Fastprep machine.

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