Team:McMasterU/Demonstrate

C. Difficile Sequencing Results


Figure 1: PCR Amplification of NAP1, NAP7, and WT TcdC: TcdC was amplified from genomic DNA of NAP1, NAP7, and wild type strains of C. difficile"


Figure 2: Sequencing Results for NAP7: NAP7 was successfully inserted into pET15b plasmid and sequenced."


Figure 3: NAP1 Sequencing Results: NAP1 was successfully inserted into pET15b plasmid and sequenced.



DNAzyme Results


Fluorophore Concentrations Optimization

  • Goal: to find the range of visible fluorescence by DNAzymes under UV light.
  • 10 uL of DNAzyme was dropped on each quadrant. Concentrations of the DNAzyme are illustrated on the images, in units of uM.
  • We've proved that our DNAzyme is functional in the fluorescence aspect and that it will show up visibly under UV light on the plate medium we hope for our prototype to be used in. We learned that the liquid DNA dropped sinks quickly into the plate's gel (composed of LB media). We also learned that liquid DNAzyme displayed in an Eppendorf tube is much more visible than DNAzyme dropped on LB medium.


Use of M9 Minimal Media vs. LB media and Optimizing Plate Medium Thicknesses

  • Goal was to find if LB medium or M9 Minimal Media was optimal for visibility of the DNAzyme on a plate-based medium, and which thickness of the gel would be most conducive for reducing background media autofluorescence interfering with DNAzyme-specific fluorescence.
  • Results: We found that M9 media was best for increased visibility of DNAzyme effects due to the increased contrast resulting from less media-originated autofluorescence. The optimal thickness of the gels for DNAzyme visibility was determined to be 1.2732 mm, with 1.088 mL poured in a small (33 mm diameter x 10 mm height) plate, and 2.5 mL poured in a larger (50 mm diameter, 10 mm height) plate.


    • Testing DNAzyme Sensitivity

    Legend

    1. (top left quadrant)E. coli K12 + DNAzyme (4.45 uL of 2.245 uM>
    2. (top right quadrant)E. coli K12 (delta RNAse, a specific strain with the RNA strand allowing E. coli ECM to bind to DNAzyme strand knocked out) + DNAzyme (4.45 uL of 2.245 uM)
    3. (bottom right quadrant)FDNA (4 uL of 2.5 uM)
    4. (bottom left quadrant)Nothing

    • Bacteria was streaked on each plate in respective quadrants and grown overnight.
    • The next morning, the DNAzymes were dropped onto the cells and the FDNA was dropped into the 3rd quadrant.
    • The M9 media is pH 8, 2.5 mL


    Fluorescence Detection Wavelength

    • Left tube: K12 + DNAzyme
    • Right tube: delta RNAse + DNAzyme

    Wavelength - Method (Filter)
    1. 473nm AttoPhos [LPB]
    2. 473nm 6-FAM [LPB]
    3. 635nm Alexa Flour 647 [LPR(ch.2)]
    4. 635nm Phosphor [IP]
    5. 473nm Alexa Flour 488 [LPB]
    6. 635nm Cy5 [LPR (ch.2)]

    Duplicates of 473 nm AttoPhos (LPB)


    473 nm 6-FAM (LPB)


    635 nm Alexa Fluor 647 (LPR(ch.2))


    635 nm Phosphor (IP)


    473 nm Alexa Fluor 488 (LPB)


    635 nm Cy5 [LPR (ch.2)]



    Baterial Autofluorescence

    • Grown for 16 hours in incubation at 37 degrees Celsius
    • Bottom row: all negative
    • Top row from left to right: 10^7, 10^5, 10^4,10^3 E.coli K12 cells


    DNAzyme Autofluorescence

    From left to right, featuring the DNAzyme quantity in picomoles per microliter: 10 pmol/5 uL, 20 pmol/5 uL, 30 pmol/5 uL, 40 pmol/5 uL



    Optimal Response of DNAzyme Fluorescence in plate without 2x Selection Buffer

    Above: pre-incubation, under natural light

    Above: after 1 hour of incubation at 37 degrees Celsius, under UV lighting

    Above: After 1 hour of incubation at 37 degrees Celsius, viewed under Typhoon imaging with 473 nm 6-FAM filter.


    Above: after 2 hours of incubation at 37 degrees Celsius, under UV lighting


    Above: After 2 hours of incubation at 37 degrees Celsius, viewed under Typhoon imaging with 473 nm 6-FAM filter.


    • DNAzyme, view counterclockwise from top left quadrant: 10 pmol, 20 pmol, 30 pmol and 40 pmol.
    • Cells concentrations dropped per quadrant: 10^7, 10^5, 10^4, and 10^3
    • Top row: Cells + DNAzyme
    • Bottom row: DNAzyme only as negative control

    DNAzyme Specificity

    With 2x Selection Buffer Added
    • Grow for 16 hours at 37 degrees Celsius
    • Cells are 10^4 cells per quadrant
    • DNAzyme: 40 pmol (dropped after bacteia has grown, incubate for 1 hour


    Tracking Fluorescence Over Time

    • DNAzyme: 40 pmol/drop (5 uL). 1 drop includes 2x SB, the other drop is without SB
    • Cells: 10^4 cells spread per quadrant and incubated for 16 hours at 37 degrees

      • Initial (before drop)


      1hr incubation


      2hr incubation