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Team:Heidelberg/Notebook/Visualization/Notebook/Largechamber
From 2008.igem.org
PURPOSE test of 1) scanning large LabTek-chamber, 2) chemotaxis against casamino acis (cas.) plug
MICROSCOPE Deltavision
STARTED 20080908
14:27, 29 October 2008 (UTC)14:27, 29 October 2008 (UTC)14:27, 29 October 2008 (UTC)14:27, 29 October 2008 (UTC)14:27, 29 October 2008 (UTC)14:27, 29 October 2008 (UTC)14:27, 29 October 2008 (UTC)14:27, 29 October 2008 (UTC)14:27, 29 October 2008 (UTC)14:27, 29 October 2008 (UTC)14:27, 29 October 2008 (UTC)14:27, 29 October 2008 (UTC)14:27, 29 October 2008 (UTC)14:27, 29 October 2008 (UTC)Marksteel 14:27, 29 October 2008 (UTC)
First Exp.
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 cylinder of PCR tube 2. 1.5 ml of M9 medium with 0.4% agar homogeneously distributed. 10 min for polymerization 3. addition of 1 ul 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 ... .. 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 14:27, 29 October 2008 (UTC)14:27, 29 October 2008 (UTC)14:27, 29 October 2008 (UTC)14:27, 29 October 2008 (UTC)14:27, 29 October 2008 (UTC)14:27, 29 October 2008 (UTC)14:27, 29 October 2008 (UTC)14:27, 29 October 2008 (UTC)14:27, 29 October 2008 (UTC)14:27, 29 October 2008 (UTC)14:27, 29 October 2008 (UTC)14:27, 29 October 2008 (UTC)14:27, 29 October 2008 (UTC)14:27, 29 October 2008 (UTC)14:27, 29 October 2008 (UTC)~ Second Exp. (2008-09-10) "BigCham": Using the HCB33_I20260 strain. Totally used chambers 2.
Preparation: I filled one chamber with 1.5ml of M9_02A and the other one with M9_04A. About half an hour later I added 1µl of the bacteria strain grown in LB, to one side of each chamber. On the other side I added 60µl M9_2A_1CA. After that 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 "Markbm" 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 will be shot. Two series for one chamber. One for the left and one for the right side. named
BigCham_20080910_HCB33_02p_8h_R
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BigCham_20080910_HCB33_04p_8h_R
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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. started: (2008-09-13) Datei 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 chambered where 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 activ 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) Datei: 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) Datei: 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
