Team:Groningen/16 August 2010

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Week 33

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

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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

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THT Staining

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SDS-PAGE

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Geeske

Double gene constructs had to be made via pSB1C3 or pSB1K3. Produced the following constructs: pSB1C3_CH, pSB1C3_dCH, pSB1C3_dCS and pSB1C3_H, but no restriction check performed yet.

Modellers: More literature research on ComXPA. Joël, Laura. Researching parts registry for information which is required to build an information standard Arend Find information about a killswitch, start to model this process Laura

Jorrit

Done sticky end ligation for Ezgi. Transformation with biofilm forming strains (Spo, Deg & Rok) according to B. sub protocol. Also done transformation with NZ8900 and ΔTasA with empty vector. no colonies were visible after 2 days for all 5 strains.

Arend Jan

Cleaned PCR product of the IS element removal attempt was blunt-end ligated. This is possible as the Phusion polymerase was used and it does not add A-overhangs to the product. PEG 4000 was added to the reaction which increases the efficiency of blunt end ligations.

- 5ul plasmid
- 2ul 10X T4 buffer
- 2ul 50% PEG4000
- 5ul T4 ligase
- 6ul MQ 

A small amount of colonies were observed after transformation. These were picked, grown, and plasmid DNA was isolated. A restriction with EcoRI and PstI revealed that the colonies had the original plasmid with the insert (gel not shown). Template DNA from the PCR must have caused this. Back to square one…