Team:Groningen/16 August 2010

From 2010.igem.org

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'''THT reference''
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'''THT reference'''
Making a reference of pure chaplins is of great importance when comparing results. For a reference 22 mg of extracted and freeze-dried celwalls of streptomyces were used. Chaplins from these cellwalls were then purified by TFA treatment. The monomerized chaplins were diluted in 1 ml demiwater. Then a variety of dilutions was made with demiwater.  
Making a reference of pure chaplins is of great importance when comparing results. For a reference 22 mg of extracted and freeze-dried celwalls of streptomyces were used. Chaplins from these cellwalls were then purified by TFA treatment. The monomerized chaplins were diluted in 1 ml demiwater. Then a variety of dilutions was made with demiwater.  
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[[Image:THT1GR.jpg]400px]
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The first expression experiment, testing all the constructs that were perhaps made so far. Tested for pellet and supernatant, raw pellets were tested as well.
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The first expression experiment, testing all the constructs that were made so far. We tested for treated pellet and supernatant, untreated pellets were tested as well.
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‘’’Exression experiment’’’
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<br>
<br>
-
Analasys was done by THT and SDS page.
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‘’’Peter & David’’’
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<br>
<br>
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For this experiment, the following B. subtilis 168 strains were used:
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<pre>
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</pre>
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<br>
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All cultures were grown overnight at 37 degrees Celsius in a shaker room.
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<br>
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Overnight cultures were  used to dilute to a B. subtilis culture of 0,1 OD, these strains were divided into ‘’induced’’ and ‘’non-induced’’. Induction with 0,5% subtilin was done at a OD of 0,5 (approximately 2,5 hours after growth of the 0,1 culture started).
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<br>
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After that the OD of the cultures was measured every .. hours.
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<br>
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‘’’Sample preperation’’’
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<br>
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After .. hours, .. after induction, the samples were collected and processed. The following procedures were used:
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<br>
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Pellet preperation ([[PelletPrepGR]])
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<br>
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Supernatant processing ([[SupernatantPrepGR]])
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<br>
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Cell disruption ([[ExtractionCellWallsGR]])
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<br>
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Lysozyme preperation ([[LysozymePrepGR]])
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<br>
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Analysis was done using SDS-PAGE ([[SDS-PAGEGR]]) and THT staining ([[THTstainingGR]]).
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<br>
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‘’’Results’’’:
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<br>
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‘’’Growth Curve’’’
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<br>
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[[Image:GrowthCurveGR1.jpg|400px]]
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<br>
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<br>
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‘’’THT Staining’’’
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<br>
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[[Image:THTGR1.jpg|400px]]
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<br>
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<br>
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‘’’SDS-PAGE’’’
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<br>
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[[Image:SDS-PAGEGR1.jpg|400px]]
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<br>
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'''Modellers:''' More literature research on ComXPA. ''Joël, Laura''. Researching parts registry for information which is required to build an information standard ''Arend''
'''Modellers:''' More literature research on ComXPA. ''Joël, Laura''. Researching parts registry for information which is required to build an information standard ''Arend''

Revision as of 15:58, 24 October 2010

iGEM Groningen 2010

Hydrophobofilm
pushing coatings into a greener future

Week 25


David


THT Staining - Chaplin ladder


THT reference

Making a reference of pure chaplins is of great importance when comparing results. For a reference 22 mg of extracted and freeze-dried celwalls of streptomyces were used. Chaplins from these cellwalls were then purified by TFA treatment. The monomerized chaplins were diluted in 1 ml demiwater. Then a variety of dilutions was made with demiwater.

Amount of diluted chaplin protein in a 250 ul sample (high concentration reference): 


100%
51,2%
25,6%
12,8%
6,4%
3,2%
1,6%
0,8%
0,4%

Blanco(demi+THT staining)

demi water



Low concentration ladder:
0,8%
0,6%
0,4%
0,3%
0,2%
blanco
demi


[[Image:THT1GR.jpg]400px]


Expression experiment - David & Peter


The first expression experiment, testing all the constructs that were made so far. We tested for treated pellet and supernatant, untreated pellets were tested as well.

‘’’Exression experiment’’’


‘’’Peter & David’’’


For this experiment, the following B. subtilis 168 strains were used:


All cultures were grown overnight at 37 degrees Celsius in a shaker room.


Overnight cultures were used to dilute to a B. subtilis culture of 0,1 OD, these strains were divided into ‘’induced’’ and ‘’non-induced’’. Induction with 0,5% subtilin was done at a OD of 0,5 (approximately 2,5 hours after growth of the 0,1 culture started).


After that the OD of the cultures was measured every .. hours.


‘’’Sample preperation’’’


After .. hours, .. after induction, the samples were collected and processed. The following procedures were used:


Pellet preperation (PelletPrepGR)


Supernatant processing (SupernatantPrepGR)


Cell disruption (ExtractionCellWallsGR)


Lysozyme preperation (LysozymePrepGR)


Analysis was done using SDS-PAGE (SDS-PAGEGR) and THT staining (THTstainingGR).


‘’’Results’’’:


‘’’Growth Curve’’’


400px




‘’’THT Staining’’’


400px




‘’’SDS-PAGE’’’


400px






Modellers: More literature research on ComXPA. Joël, Laura. Researching parts registry for information which is required to build an information standard Arend


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