Team:CMUQ/Notebook

Notebook

  • Sunday 7/2

    Dina, Najla and Maya discussed lab space, reagents and work time, as well as future meetings:

    • Main Lab Space: 2033
    • Days and Times: based on experiments and is specific to each group, need to inform Bernadette beforehand
    • Regents: all reagents needed for the next two weeks are available, need to know more about SRB.
    • iGEM group meetings will be held on Sundays: update logs during the weekend, present work on Sunday for the whole team

  • Monday 7/3

    Dina:

    • Email Dr. Vincent with updates
    • Re-read protocols, and write them by hand into lab notebook for next day

  • Tuesday 7/4

    Dina:

    • Prepared LB-Agar Chloramphenicol plates
    • Protocol:
    • Work on presentation for tomorrow's team meeting

  • Wednesday 7/5

    iGEM team meeting

    Interlab:

    • Obtained DH5α cells (Genetics Lab 2013) from Maya
    • Start preparing competent DH5α cells:

  • Thurday 7/6

    Interlab

    • Continue preparing competent DH5α cells
    • Obtain regents needed for next week preparation of competent cells
      • MgCl2 (third floor lab)
      • CaCl2 (2033 cab)
      • ddH2O (Maria, Chemlab)
      • Glycerol (2033 Flammable cab)

  • Sunday 7/9

    DspB Lab:

    • Meeting with Dr. Vincent: changed protocol for the preparation of competent cells as Cheryl mentioned in her email, need to use ultra competent cells (from Dr. Ihab's lab) → will start next week

    Biofilm Lab:

    • Meeting with Bernadette and Maya to discuss materials and applinces being used in the lab for Biofilm Production.
    • Meeting with Professor Vincent to discuss how to proceed with porject:
      • 96 well plates.
      • Add samples of SRB directly onto the wells (no inoculation); see if media is required for biofilm formation.
      • Possible simulation using appratus in lab (Bioflux 2000; https://bioflux.fluxionbio.com/microbiology )

    To do:

    • Write up protocol for biofilm production on plastic pipe.
    • SRB media recipe (just in case wemay need to use it)
    • Appratus set up

  • Monday 7/10

    Interlab:

    • Continue preparing competent DH5α cells
    • Preparation of MgCl2 and CaCl2 solutions for CaCl2 competent cells preparation
    • LB media preparation

  • Tuesday 7/11

    Interlab:

    • Finish preparing competent DH5α cells
    • Prepare CaCl2 w/ Glycerol solution
    • Filter sterilize all solutions

    Group meeting:

    • Test for DspB secretion outside after successful transformation

    Skype meeting with Chyrl

  • Wednesday 7/12

    Biofilm lab:

    • LB Apmicilin/Arabinose plates were made.

    Interlab:

    • TPrepare SOC media for competency test (Obtained SOC from Bernadette, didn't make our own)
    • Competent cell test using Kit

    Skype meeting with Pittsburgh for collaboration

  • Thursday 7/13

    Interlab:

    • Competent cells test: there was no growth on any of the plates except for tiny colonies on the 100 pb/µL plate. Another method to prepare competent DH5α cells should be utilized.

    Meeting to discuss Wiki with Yasmin

    Biofilm lab:

    • Plated flouresent DH5α were incoulated on LB Ampicilin/Arabinose broth on 50 ml kleftt flask; left over 2 days in shaker at 37°C.

  • Sunday 7/16

    Biofilm lab:

    • Using spechtophotometer, OD of inoculated cultre was found to be ~ 375. 3 dilutions were made at 7.5X, 13X and 26X. Dilutions and culture were left to incubate overnight in the air shaker at 37°C.

  • Monday 7/17

    DspB Lab:

    • Obtain cells from Dr. Ihab lab, alongside the protocol
    • Cells from competent cell test grew after ≈ 5 days

    Biofilm Lab:

    • Dilutions and original culture in broth were checked under UV and no fluorescence was found. Plated GFP-DH5α cells were found to be fluorescent. Cultures in broth were scrapped and new LB Ampicilin/Arabinose broth was planned to be made.

  • Tuesday 7/14

    Biofilm Lab:

    • LB Ampicilin/Arabinose broth was made.

    DspB Lab:

    • Prepared more Chl LB plates for transformations

  • Sunday 7/23

    DspB Lab:

    • Transform DspB (BBa_K1659200 | Kit plate 7 | well 15C) into ultra competent cells

    Biofilm Lab:

    • Inoculate GFP-DH5α cells in 50 ml LB Amp/Arab broth in 150 ml Kelft flask. Left in shaker at 37 overnight to culture.

  • Tuesday 7/25

    DspB Lab:

    • Transformation of DspB (BBa_K1659200 | Kit plate 7 | well 15C) into ultra competent cells.
    • Result: three colonies observed on LB Chl plate, no red fluorescence.

    Biofilm Lab:

    • Dilute by 10-fold GFP-DH5α culture and plate in 96 well plate. Left to incubate over 2 days.

  • Sunday 7/16

    Biofilm lab:

    • Using spechtophotometer, OD of inoculated cultre was found to be ~ 375. 3 dilutions were made at 7.5X, 13X and 26X. Dilutions and culture were left to incubate overnight in the air shaker at 37°C.

  • Thursday 7/27

    Biofilm Lab:

    • GFP-DH5α biofilm were shown on 96 well plate.

  • Sunday 7/30

    DspB Lab:

    • Overnight culture of DspB plate (from fridge) into LB Chl media, picked two colonies and left overnight shaking at 37˚C.

  • Monday 7/31

    DspB Lab:

    • Mini prep to extract DspB (BBa_K1659200 | Kit plate 7 | well 15C) plasmid DNA.
    • Agarose mini-gel run of DspB plasmid to check size: successful transformation as the size was ≈ 3Kb as expected for DspB plasmid

  • Tuesday 8/15

    DspB Lab:

    • Plated the remaining ultra competent cells ≈ 20 µLon LB plate to make more ultra-competent cells
    • Plated 50 µL of Saad's prepared DH5 Alpha on LB plate to make more competent cells
    • Transformation of DspB-DspA (BBa_K1659211 | Kit plate 7 | well 15I ) into LB-Chl plate
    • Need to do this for gene expression: DspB-DspA contained in the pSB1C3 backbone, was extracted via Miniprep. The NcoI restriction site was introduced upstream of each of our gene sequences (except BBa_K1659501 and BBa_K1659601) via PCR to facilitate their insertion into the arabinose-inducible pBAD/HisB commercial expression vector, and the insert-containing expression vectors were subsequently cloned separately into the standard laboratory E. coli K-12 strain MG1655 as well as a chemotaxis knockout strain E. coli RP437 ∆FliC.

  • Wednesday 8/16

    DspB Lab:

    • Ultracompetent cells growth, as well as DH5 Alpha cells.
    • Plate with transformed DspB-DspA (BBa_K1659211 | Kit plate 7 | well 15I) didn't show any colonies, will keep in incubator for longer

  • Thursday 8/17

    DspB Lab:

    • Growth on the DspB-DspA (BBa_K1659211 | Kit plate 7 | well 15I) plate, only 4 colonies, leave over weekend
    • Store other plates (cells) in the fridge

  • Monday 8/21

    DspB Lab:

    • Overnight culture of DspB-DspA plate (from fridge) into LB Chl media, picked two colonies and left overnight shaking at 37˚C.

    Yasmin and Dina:

    • Lab Photoshoot for Wiki

    Saad and Dina:

    • Discuss SRB tagetting, logo and abstract

  • Tuesday 8/22

    DspB Lab:

    • Mini prep to extract DspB-DspA (BBa_K1659211 | Kit plate 7 | well 15I)
    • Agarosegel run of DspB-DspA unsuccessful

    iGEM Team meeting

  • Wednesday 8/23

    DspB Lab:

    • Individual meeting with Cheryl

  • Sunday 9/3

    Dina:

    • Meeting with Oxy Qatar for human resources

  • Tuesday 9/19

    DNA sequences arrived from IDT

  • Friday 8/29

    Interlab Study submission

  • Thursday 10/5

    Photoshoot

    Team meeting

    Wiki updates

    Transform pRSET emGFP into DH5 Alpha (max comp)

  • Sunday 10/8

    Dina and Albandari:

    Dina and Kawthar:

    • Double digest of pRSET plasmid with EcoRI and BamHI successful

    Aisha:

    • Digestion of 3 samples of DspB DspA plasmid with PstI to check size

  • Monday 10/9

    Aisha and Albandari:

    • Transform pSB1C3 plasmid from kit (we will use for salt sensor)

    Dina and Alreem:

    • Agarose gel run with pRSET digest to extract, undigested plasmids to check size
    • Agarose gel run with DspB -DspA digested (BBa_K1659211, BBa_K1659201, and Dina's prepared BBa_K1659211)

    Saad:

    • Gel imaging

  • Tuesday 10/10

    Saad:

    • Extraction of pRSET plasmid from gel: concentration = 19.8 µg/mL

    Dina and Alreem:

    • PCR of DspB DspA

  • Tuesday 10/11

    DspB DspA:

    • Cleanup PCR products DspB DspA samples and combine samples
    • Digestion of PCR products with EcoRI and BamHI
    • Ran a gel to check size
    • Cleanup
    • Ligation into pRSET plasmid with instant sticky-end ligase mix NEB
    • Transformation

    gBLOCK:

    • PCR samples

    gBLOCK 1: WT proU osmolarity promoter

    gBLOCK 2: proU-B0034-mRFP-B001

    gBLOCK 3: Consensus proU osmolarity promoter

    gBLOCK 4: WT proU-RBS-mRFP-B0015

  • Thursday 10/12

    PCR cleanup

    Digestion

    Cleanup

    Ligation

    Transformation

  • Sunday 10/15

    There was growth of pRSET em-GFP plasmid with insert DspB DspA

  • Monday 10/16

    DspBDspA in pRSET:

    • Mini prep
    • Transform in BL21

    ProU:

    • Inoculate into LB-Chl