Team:Heidelberg/Notebook/Visualization/Notebook/Chemotaxis

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Test of chemotactic activity

STARTED 2008-09-08

PURPOSE:
Test of 1) chemotactic activities of HCB33 and MG1655 depending on special strain abilities and the medium (LB, TB and M9) (due to a visible lack of chemotactic activities during our M9-A measurments), 2) developing a method visualizing movin' bacteria

MICROSCOPE:
Deltavision

STRAIN:
HCB33 and MG1655 with plasmid BBa_I20260 in pSB3K3 (reference promoter, measurement kit, 2008.igem.org/measurement)

- Using the small 2x8 microscope chamber.

METHOD:

Every chamber will be filled with 200µl of the appropriate Medium.
We are using LB, TB and M9.

Then depending on the cells densitiy 1-2µl of a cell sample is added to the center of a single chamber.

The prepared chambers can now be analysed under the microscope.
Taken time lapsing pictures can later be used to produce a videofile of the cell's movement.

First Screening: (2008-09-09)

Two overnight cultures (one MG1655 the other one HCB33) in LB.
Two further overnight cultures of the same strains in M9.

Each of the LB cultures brought into a chamber with 200µl LB.
And each of the M9 cultures was brought into a chamber with 200µl M9.

Furthermore HCB33 from LB was put into a chamber with M9 and HCB33 from M9 was set into a chamber with LB.

Totally 6 chambers used.

¦ ¦ ¦ ?
¦ ¦ ¦ ?

First Results:
- Strains from the LB overnight cultures were really bouncy. With HCB33 some chemotactic-like movements could be spectated. Even the HCB33 LB culture put into the M9 Media was really active.
- Strains from the M9 media were obviously inactive. Few shivering movements could be watched. A few more after putting the HCB33 strain from M9 to LB. But still just a few.

lanned second Screening (2008-09-10)

Puttin LB cultures either HCB33 or MG1655 in one of the appropriate media. (LB, TB or M9) -> 6 chambers Also testing one HCB33 and MG1655 NATIVE strain in LB for differences in chemotactic activities compared to the I20260 strains.

Totally all 8 chambers will be used.

     LB  TB  M9  LB(natives)

HCB33 ¦ ¦ ¦ ¦
MG1655¦ ¦ ¦ ¦

update* At the beginning TB wasn't available. So the first analysis was taken without a TB probe.

This time really active Bacteria could be observed. Maybe they're more activ, if the overnight culture is taken from a glycerol stock.

Later the experiment was repeated with TB and the other Media incubating the bacteria for about 2 hours. Called "2nd Encounter". Because a longer incubation time might be needed to get any effects of the media on the chemotactic activity of the bacteria.

This time again 6 chambers will be used. There is no need of testing the native strains again against its I20260 version the first results of "Impact" should be enough to figure out if there is a nameable difference between the two strain versions.

Third experiment (2008-09-13)

Observation of chemotaxis

Actors: HCB33 and MG1655 I20260 LB overnight cultures
Using small chambers. Testing LB 0.25A, LB 05A ,TB 0.4A and M9 0.4 A media. Each for both strains.

     LB02LB05TB04M904

HCB33 ¦ ¦ ¦ ¦
MG1655¦ ¦ ¦ ¦

Results: HCB33:

LB025 At the beginning really active, many chemotactic like movements/ 3 hours latrer slightly less active, but still a few moving bacteria / after the addition of casamino acids active, many chemotactic like movements.
LB05 really inactive / 3 hours later only few moving bacteria / after the addition of casamino acids still not active
TB04 generally active, but just shivering / 3 hours later much more active, many chemotactic like movements / after the addition of casamino acids enhanced activity, many chemotactic lie movements
M904 rather inactive, no straight movements / 3 hours later slightly more active, but not straight / after the addition of casamino acids no change

MG1655:

LB025 schwach aktiv / 3h später stark verminderte Aktivitaet / +CA aktiv, aber kaum direkte Bewegungen LB05 sehr inaktiv / 3h später keine bzw. max. sehr geringe aktivitaet beobachtbar / +CA kaum aktiv TB04 aktiv / 3h später sehr aktiv, nicht so viel direkte bewegung / +CA keine Änderung M904 eher inaktiv, nicht geradlinig / 3 h später leicht aktiv, aber nicht direkt / keine Änderung

-> HCB in TB zeigte die bemerkenswertesten Reaktionen. TB seems to enhance chemotactic acticity at least for HCB33.

Each chamber was filled with 200µl of the appropriate media. After 15 minutes bacteria strains have been added and distributed through the whole chamber.

- First measurment after 0 hours - Second measurment after 3 hours - Third measurment after 3 hours + centered 5µl of 10% casamino acid solution. (- Fourth measurment after 4 hours looking for higher cell density at the casamino acid spot)

IV. Experiment (2008-09-15)

HCB33 and MG1655 TB overnight cultures Each of the used chambers was filled with 200µl of the appropriate media, 1 µl TB overnight culture was added and spread. The whole experiment is similiar to (2008-09-10 2nd). Instead of LB overnight cultures TB overnight cultures were used.

200µl media 1µl culture

      LB  TB  M9

HCB33 ¦ ¦ ¦ MG1655 ¦ ¦ ¦

Beobachtung: HCB33: LB 1h sehr aktiv /4h aktiv einige direkte Bewegungen TB 1h sehr aktiv /4h einige direkte M9 1h viele schlapp, wenige überhaupt aktiv /4h eher inaktiv, wenige direkte

MG1655: LB 1h schlapp /4h ein wenig aktiv, nicht direkt TB 1h schlapp /4h dicht gewachsen, nicht gerichtet, zittrig M9 1h inaktiv /4h kaum aktiv, nicht direkt

No difference after the addition of 10% CA solution after 4 hours!!!

Result: HCB33 is heavily more activ when incubated over night in TB. Also HCB33 is generally more activ incuated at TB than MG1655.

V. Experiment (2008-09-17)

Measurment of HCB33 and MG1655 on grown on LB at an OD of 0.5. Therefore I incubated the strains on LB overnight and diluted the cultures on the next morning to an OD less than 0.5 and let them incubate for 1 hour.

This experiment was used to proof the chemotactic ability of LB overnight cultures, but it also used as a presentation for the Prometheus Tv Team.

->Result. LB is as well as TB an apropriate media. HCB33 activ MG1655 generally inactiv.

<> Furthermore only HCB33 will be used.

VI. Experiment (2008-09-22)

<> Video files of the BigCham experiment 2008-09-22_HCB33_TB&Teth_Plugged_16h showing a spot at the right and a spot at the left side of the chamber.

<> TB and Teth media were used to test the usability of Tethering Buffer in chemotaxis assays. Also HCB33 Teth overnight cultures were used.

200µl media 3µl of TB culture, 6µl of Tethovernight culture

           TB  Teth

HCB33 ¦ ¦ HCB33(Teth) ¦ ¦

Beobachtung: HCB33: TB beeindruckend aktiv Teth sehr inaktiv /CA einige aktiv

HCB(Teth): TB sehr inaktiv /CA keine signifikante Änderung TB sehr inaktiv /CA keine signifikante Änderung