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Team:Groningen/16 August 2010
From 2010.igem.org
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
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
THT Staining
SDS-PAGE
Modellers: More literature research on ComXPA. Joël, Laura. Researching parts registry for information which is required to build an information standard Arend
