Team:Heidelberg/Notebook/Visualization/Notebook/Largechamber

From 2008.igem.org

Latest revision as of 19:49, 29 October 2008

Large chamber tests

STARTED: 2008-09-08

PURPOSE:
Test of 1) scanning large LabTek-chambers, 2) chemotaxis against casamino acids and casamino acid plugs

MICROSCOPE:
Deltavision

A few pictures which have been taken during these experiments can be viewed under Projects.

First Experiment

STRAIN:
MG1655 with plasmid BBa_I20260 in pSB3K3 (reference promoter, measurement kit, 2008.igem.org/measurement) inoculation from dense (OD=1.0? (estimated)) culture.

PREPARATION OF CHAMBER:
1. Plug with casamino acids: 50 ul of M9 medium with 2% Agar and 1% Cas. in a PCR tube cylinder.
2. 1.5 ml of M9 media with 0.4% agar homogeneously distributed. 10 min for polymerization.
3. Addition of 1 µl bacterial suspension.
4. 1.5 ml of M9 medium with 2.0% agar homogeneously distributed. 10 min for polymerization.

GRID:
Definition of node grid Markbig containing 6x8 nodes.

1 7 ... 43
2 8 ... 44
3 9 ... 45
4 10 .. 46
5 11 .. 47
6 12 .. 48

MEASUREMENTS:
On 20080908 in folder 20080908_MG1655_bigR_04p_0h:
Measurement of right half (around bacterial startin point)
On 20080909 in folder 20080909MG1655_BIG_04p_16h:
Measurement of right and left half of chamber.

RESULTS:
1) Scanning of chamber possible, no collision of chamber with frame.
2) no accumulation of bacteria around casamino acids plug on second day, 20080909.
3) no chemotactic runs could be observed. most bacteria inert, only some tumbling.
4) large cell plaques on second day, 20080909.

Second Experiment (2008-09-10)

"BigCham":
Using the HCB33_I20260 strain.
Totally used chambers 2.

Preparation:
One chamber was filled with with 1.5ml of M9_02A and the other one with M9_04A.
About half an hour later 1µl of the bacteria strain grown in LB was added to one side of each chamber. On the other side 60µl M9_2A_1CA were spotted.
Afterwards the whole chamber was filled with additional 1,5ml of M9_2A

-> !!! problem: Neither the 04p_A nor the 02p_A stiffens fast enough.
So in most cases the first layergets mixed with the 2p_A (second layer). This could also be a problem for the bacteria (heat and too high agar percentage media).
This will be discussed during the Teammeeting on friday 12.09.2008.

Furthermore two series were shot each serie shows the right half of one of the chambers: Using the same shape of the frame, but a modyfied version called which is a bit more lower.

Series were named:
BigCham_20080910_HCB33_02p_0h_R
BigCham_20080910_HCB33_04p_0h_R

Chambers incubated at 37°C for 7-8 hours
Additional 4 series were shot. Two series for one chamber. One for the left and one for the right side named:
BigCham_20080910_HCB33_02p_8h_R
""""""""""""""""""""""""""""""L
BigCham_20080910_HCB33_04p_8h_R
""""""""""""""""""""""""""""""L

Results: Nothing really usable. Bacteria didn't move.
We figuered out that HCB33 overnight cultivated at 37°C has the highest chemotactic activity.
Furthermore for the next experiments HCB33 TB overnight cultures will be used.

III. Experiment started: (2008-09-13)

Data: 2009-09-15

Changes:
- Second layer declared sensless.
- HCB33 TB overnight highest activities.
- 100µl casamino acid plugs

2 chambers were each filled with 3ml TB02, one 100 µl plug of M9 2A 1CA was added to one side of one chamber, to each center of a chamber 1 µl LB overnight culture of HCB33 was added.

These chamberes were incubated from saturday evening to monday morning at RT. -> Too long
Bacteria where allready distributed over the whole chamber. -> Repeat Pictures have been taken.
Pictures from (2008-09-15 HCB33LB)
Results: Generally high active bacteria, even after such a long time.

The same experiment. Only change two HCB33 TB overnight cultures were used instead of two LB overnights.
This time a a first measurment will be taken after a few hours and a second on the following day.

IV. Experiment (2008-09-19)

Data: 2008-09-20

2ml Teth02
100µl M9 2A 1CA plug
3µl HCB33 (1:1 purified from TB overnight with Tethering Buffer (4000rpm 10min))

Steps: Plug -> media -> bacteria
Bacteria are set into the center of the chamber and the plug on the left side.

Several series have been taken with OAI, changing the exposure:
0.1%
1%
10%
32%

-> Results: OAI is a good solution for the quantification of chemotaxis. Intensity measurments.

V. Experiment (2008-09-21)

Data: 2008-09-22

2ml Teth02A and TB02A.
100µl M9 2A 1CA Plug each.
3 µl bacteria TB overnight culture each

Pictures have been taken with DV via OAI at an exposure of 0.1% with minumum exposure duration 8-10 seconds.
Thickness ~70µm

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