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    Week 23
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    Week 23

    Tuesday, 6/8/2021Wednesday, 6/9/2021Table 1. Absorbance of E.coli culture in shaking conditionsTable 2. Sampling timetable for Curli testingTable 3. Sample 1 in Curli testing experiment after 2h inoculatedTable 4. Sample 2 in Curli testing experiment after 9.5h inoculatedThursday, 6/10/2021Table 5. Sample 3 in Curli testing experiment after 21.5h inoculationTable 6. Sample 4 in Curli testing experiment after 33.5h inoculationFriday, 6/11/2021Table 7. Sample 5 in Curli testing experiment after 45.5h inoculationTable 7. Sample 6 in Curli testing experiment after 57.5h inoculation
    Tuesday, 6/8/2021
    Preparation of solutions
    Goal: prepare solutions for E.coli (LB, PBS)
    ●
    LB broth was prepared following protocol in ​LB medium recipe.
    ○
    Triptone (kurs lab) prod. Nº T1332.0500. Exp. date. 31/08/2021
    ○
    NaCl (kurs lab) prod. Nº 1.06404.1000. Exp. date. ?
    ○
    Yeast extract (kurs lab) prod. Nº 1.03753.0500. Exp. date. 05/06/2021
    ●
    PBS was prepared following protocol in ​PBS solution recipe.
    ○
    PBS 10X → 500 mL
    ■
    Na2HPO4 (kurs lab) prod. Nº. 106586.0500. Exp.date. 31/07/2020
    ○
    PBS 1X → 1000 mL (Diluting PBS 10X with dH2O 10 times)
    ○
    PBS 1X + 2% SDS + 1% triton x-100 → 1000 mL
    ■
    SDS (kurs lab) prod. Nº. 102918. Exp. date. ?
    ■
    Triton x-100 (kurs lab) prod. Nº. 9002-93-1. Exp. date. ?
    Distilled water from the lab tap was used in all solutions.
    PBS 1X + 2% SDS + 1% triton X-100 was kept mixing with a magnetic flea at 80ºC for 30 min approx.
    All solutions were kept at the kurs lab at room temperature.
    OBS! KCl and KH2PO4 were taken from the department. In LB medium, step 4 is missing, pH was not adjusted to 7.0.
    ●
    Thioflavin T solution was prepared following protocol in ​Thioflavin T (1mM) .
    ○
    After filtering the ThT solution, it was aliquoted in 1mL eppendorf tubes and stored in the freezer (course lab).
    OBS! All solutions containing PBS precipitate after autoclave. Must repeat with cleaner bottles.
    Wednesday, 6/9/2021
    E.coli MC4100 cultivation
    Goal: cultivate E.coli to make a stock.
    1.
    To make a work cell bank, the cells from Gothenburg are taken, stored in agar (white envelope in the fridge in kurs lab).
    2.
    To inoculate the broth protocol ​Growth E.coli MC4100 Aerobic conditions was followed.
    3.
    Instead of 1 mL, some colonies are taken.
    4.
    2 falcon tubes x 50 mL were incubated in shaking for 9.5h.
    Mean OD = 1.11
    OBS! Cells are growing ok with shaking. These samples are discarded and another experiment will be performed to do the work cell bank.
    Curli testing
    Goal: cultivate E.coli to test Curli production.
    6 falcon tubes x 50 mL LB are inoculated with E.coli MC4100 colonies from the plate, following protocol ​Growth E.coli MC4100 Aerobic conditions.
    All tubes are inoculated at 8.30 and kept in the oven at 37ºC (oven in the cours lab).
    Sampling:
    In all experiments/samples 2 different ThT concentrations are tested (10 µM and 40 µM). Protocol ​Growth E.coli MC4100 Aerobic conditions was followed in all samples.
    SAMPLE 1. 10.30 (2h)
    From the culture, 6 mL were taken.
    ●
    3 mL (triplicates) cells + broth to measure absorbance and fluorescence at 10 µM ThT concentration.
    ●
    3 mL (triplicates) cells + broth to measure absorbance and fluorescence at 40 µM ThT concentration.
    Abs. at 600 nm (BLANK: LB)
    Store supernatant in the cold room.
    OBS! Centrifugation in step 12 was made at 4ºC instead of RT. In Agilent Cary Eclipse a method was set up with the following parameters:
    Scan set up → Emission:
    ●
    Excitation (nm): 450.00
    ●
    Start (nm): 455.00
    ●
    Stop (nm): 600.00
    Scan control: Medium
    Multicell holder
    SAMPLE 2. 20.00 (9.5h)
    Abs at 600 nm (BLANK: LB). Espectro. WPA nº6 (kurs lab).
    Store supernatant in the cold room.
    OBS! Centrifugation in Step 12 was made at 25ºC = RT. Same parameters as sample 1.
    Thursday, 6/10/2021
    SAMPLE 3. 8.00 (21.5h)
    Abs at 600 nm (BLANK: LB). Espectro. WPA nº6 (kurs lab).
    Store supernatant in the cold room.
    OBS! Fluorescence measurement for 40 µM ThT repeated (-2 mL supernatant).
    SAMPLE 4. 20.00 (33.5h)
    Abs at 600 nm (BLANK: LB). Espectro. WPA nº6 (kurs lab).
    Store supernatant in the cold room.
    OBS! All the sampling and removing the pellet was made under sterile conditions (none of the previous samples were taken under sterile conditions).
    Friday, 6/11/2021
    SAMPLE 5. 8.00 (45.5h)
    Store the supernatant in the cold room.
    SAMPLE 6. 20.00 (57.5h)
    Store the supernatant in the cold room.
    Preparation of LB agar plates
    Goal: prepare LB agar plates to test pellets from samples 4, 5 and 6. If they grow, the cells were not broken during sonication, if no-growth → dead cells, may repeat the experiment of Curli testing.
    1.
    Prepare LB agar following protocol ​LB agar plates.
    2.
    Autoclave 200mL LB agar + 1L LB broth (new) + 1L PBS + 2% SDS + 1% Triton-X100.
    3.
    12 plates are made with the 200 mL agar, all plates are sterilized and handed under the bunsen burned.
    4.
    Pellets of samples 4, 5 and 6, are resuspended in PBS and plated. For each sample 2 plates are made, one with 1mL of sample and one with 0.5mL.
    5.
    Plates are kept  in the oven for the weekend.
    6.
    Growth is observed in all plates, even though no colonies are differentiated due to the high amount of cells.
    7.
    From one of the plates, some colonies are taken and cultivated in a falcon tube with 50mL of liquid LB broth.

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