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From 2010.igem.org

Assembly of biobricks in pSB1C3-backbone for submission to parts registry by Maarten

Control restrictions with EcoRI and PstI on constructs:

2 µL construct

2 µL buffer O

0,5 µL EcoRI

0,5 µL PstI

15 µL MQ

Construct	Expected band sizes (bp)
pSB1C3-C	2031, 848
pSB1C3-DC	2031, 872
pSB1C3-E	2031, 317
pSB1C3-H	2031, 302
pSB1C3-Sortase	2031, 689
pSB1C3-RFP	2031, 1069

The gel that was run did not show any DNA present on the gel. Experiment was continued on 8 September.

Expression experiments -David & Peter

Two expression experiments were done this week.

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, the appropriate antibiotics were used at all points in time during this experiment.

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

400px

SDS-PAGE

400px

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, the appropriate antibiotics were used at all points in time during this experiment.

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

400px

Continous induction of chaplins

As a follow up experiment for the last biofilm induction Bacillus subtilis wit Chaplin E and chaplin H were grown while continuously adding subtilin to keep expression up. After 3hrs of growth they where induced with 1%subtilin. Every hour an additional 0.5%subtilin was added. As a negative control uninduced strains where used. Furthermore shaking cultures (induced and uninduced) where grown simultaneously to check OD levels. Only the chaplin E strain showed reduced growth, but no differences other than retarded growth were visible.

Modellers: Work on gene expression model Joël, Djoke, Laura