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<div class="row"><p><i>Procedure is a modified form of NCBI’s Microtiter Dish Biofilm Formation Assay <a>(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182663/)</a></i></p></div>
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<div class="row"><p><i>Procedure is a modified form of NCBI’s Microtiter Dish Biofilm Formation Assay <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182663/">(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182663/)</a></i></p></div>
  
 
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Latest revision as of 22:07, 15 October 2018

Experiments

Transformations

BI21 Cells:

  1. Thaw 50 µL vial of BL21 cells on ice
  2. Inoculate 2µL DNA into 50µL BL21 cells
    • incubate on ice for 30 minutes
    • heat shock at 42℃ for 10 seconds
    • incubate on ice for 5m
  3. Inoculate 250µL LB into vial
    • incubate at 37℃ with shaking for 1.5 hours
  4. Plate 1x/9x

JM109 Cells:

  1. Thaw vial of JM109 cells on ice
  2. Label 14 mL Falcon tube & plane on ice
  3. Inoculate 50 µL JM109 cells into Falcon tube
  4. Inoculate 2µL DNA into 50µL JM109 cells
    • incubate on ice for 20m
    • heat shock at 42℃ for 45 seconds
  5. Inoculate 950µL LB into vial
    • incubate at 37℃ with shaking for 1.5 hours
  6. Plate 1x/9x


Electrophoresis/Gel Protocols

1% Agarose Electrophoresis Gel

  1. Add 0.35 g agarose to 35 mL 1x TAE in 100mL conical flask
  2. Heat in microwave for 35 seconds
  3. Swirl and heat in microwave for 25 seconds
  4. Swirl and heat in microwave for 15 seconds
  5. Swirl and heat in microwave for 7 seconds
  6. Swirl and ensure no ‘floaties’
  7. Cool outside of conical flask with water until ‘hand not’
  8. Pour into gel mold and add well comb

Gel Extraction

  1. Use ethanol-wiped scalpel to excise appropriate bands
  2. Mix and incubate at 50°C for 10 min
    • Vortex every 2 mins until gel slice is completely dissolved
  3. Place nucleospin column into 2mL collection tube
    • Add sample
    • Centrifuge at 11,000 xg for 1 minute
    • Discard throughflow and place column back in same tube
  4. Add 700 mL Buffer NT3
    • Centrifuge at 11,000 xg for 1 minute
    • Discard throughflow and place column back in same tube
  5. Repeat step 4
  6. Centrifuge at 11,000 xg for another minute
    • To remove buffer
      • Discard throughflow
      • Centrifuge at 11,00 xg for add minute
      • Place column in a clean, labelled 1.5 mL tube
  7. Add 25 mL prewarmed (50°C) Buffer NE
    • Incubate at 50°C for 5 min
    • Centrifuge at 50 xg for 1 minute
    • Centrifuge at 11,000 xg for 1 minute

Plasmid Transformation Protocol

  1. Aliquot 1 ml media into 1.5 mL tubes
    • Place in 42°C waterbath
  2. Prechill labelled 14 mL round-bottom falcon tubes
    • Add mL cell culture to tube
    • Add appropriate amount of ligase rxh (2-2.5 L)
    • Incubate in nice for 20 minutes
  3. Heat shock cells by placing at 42℃ for 45 seconds in waterbath
    • Place on ice for 2 minutes
  4. Add 950 mL preheated media to each tube
    • Incubate at 37℃ with 215 rpm shaking for 1.5 hr -> 20 minutes

Plating

  1. Plate on LB-antibiotic plates
    • Plate 100 ml for 1x transformation
    • Plate 100 mL 9x centrifuge remaining transformation at 2000 xg for 5 minutes and resuspended pellet in 100 L media

Plates Protocol

  1. Follow LB Agar Recipe
    • Make sure to put stirring bar in water
    • Needed to make into homogenous gel
    • Needs to be sterile
  2. Put into Autoclave
    • Lid should not be too tight
  3. Put in ice bath 42°C
    • Cool till not burning hand
    • Put on stirring plate
  4. Put sterilized plates in Lamenia shield
  5. Using micropipette put .5 mL of chlor. And amp. agar solution
    • 1000x dilution
    • Amp. has to be defrosted in dark drawer
  6. Pour solution into plates
    • Thin layer fill bottom
    • Take lid slightly off to prevent condensation

QIA Prep Spin Mini Prep Kit

  1. Example for 18 mL LB add
    • 18 L20 mg/ L Kanamycin (antibiotic)
    • 18 L 50 mg/ L ampicillin
  2. Aliquot 3.5 mL into labelled round bottom falcon tubes
  3. Using sterile toothpicks- pick a single colony forming unit (cfu) and inoculate tube
    • Incubate overnight at 37°C with 215 rpm shaking
  4. To 100 mL 60% glycerol stock, add 300mL overnight culture to appropriately labelled tube
    • Store at -80°C
  5. Pellet remaining cells by centrifugation in 1.5 mL tube at 13,2000 rpm for 1 min
    • Discard supernatant and repeat
  6. Resuspend pellet in Buffer P1 250 mL
    • Add 250 L Buffer, P2 and mix by inverting 10x
    • Add 350 L Buffer N3 and immediately mix by inverting 10x
  7. Centrifuge at 13, 200 rpm for 10 minute
  8. Pipet supernatant into labelled QIA prep spin column
    • Centrifuge at 13, 200 rpm for 1 minute
    • Discard thoroughflow
  9. Wash column by adding .5 mL Buffer PB *regular for low copy number plasmids*
    • Centrifuge at 13, 200 rpm for 1 minute
    • Discard thoroughflow
  10. Wash by adding .75 mL Buffer PE
    • Centrifuge at 13, 200 rpm for 1 minute
    • Discard thoroughflow
    • Centrifuge for an addition minute to ensure removal of residual wash buffer
  11. Place spin column in a clean, labelled 1.5 mL tube
    • Add 50 LEB to center of column & let stand for 1 minute at room temperature
    • Centrifuge at 12,000 rpm for 1 minute
    • Store at -20℃

Making Motility Plates

  1. Follow Motility Plate Recipe
    • Recipe
      • 5 grams of Tryptone
      • 2.5 grams of NaCl
      • 1.25 grams of Agar
      • 500 mL H2O
    • Make sure to put stirring bar in water
    • Needed to make into homogenous gel
    • Needs to be sterile
  2. Put into Autoclave
    • Lid should not be too tight
  3. Put in water bath 42°C
    • Cool till not burning hand
    • Put on stirring plate
  4. Put sterilized plates in Lamenia shield
  5. Pour solution into plates
    • Thin layer fill bottom
    • Take lid slightly off to prevent condensation

Making TSA Plates

  1. Follow TSA Plate Recipe
    • Recipe
      • 30 g of TSB
      • 15 g of Agar
      • 1 Liter of H20 18 mΩ
    • Make sure to put stirring bar in water
    • Needed to make into homogenous gel
    • Needs to be sterile
  2. Put into Autoclave
    • Lid should not be too tight
  3. Put in water bath 42°C
    • Cool till not burning hand
    • Put on stirring plate
  4. Put sterilized plates in Lamenia shield
  5. Pour solution into plates
    • Thin layer fill bottom
    • Take lid slightly off to prevent condensation

Yarrowia Testing

Growing Yarrowia Culture

  1. Pipette 5 mL of TSB into a culture tube
  2. Use a wand to swipe a colony of Yarrowia from an already grown plate of Yarrowia
  3. Place wand with colony of Yarrowia into culture tube
    • Flick wand for 15 seconds
  4. Incubate overnight at 27°C with 200 rpm shaking

Preparing Chitinase Testing Enzyme

  1. Measure out 2 g of chitinase from Streptomyces Griseus
    • Place into 2 mL microcentrifuge tube
  2. Resuspend chitinase
    • Pipette 0.100 mL of PBS into tube
    • Vortex tube for 15 seconds
  3. Store in -20°C freezer

Chitinase Testing Against Yarrowia

  1. Dilute Yarrowia culture 1:000
    • Use 1.5 mL microcentrifuge tube
      • Pipette .999 mL of TSB into tube
      • Pipette .001 mL of grown Yarrowia culture into tube
      • Inoculate tube by flicking 4-6 times
  2. Pipette .300 mL of diluted Yarrowia culture on TSA plate
    • Use L-spreader to spread diluted Yarrowia culture
    • Let plates dry in fume hood for 30 minutes
  3. Perform steps 4-6 a total of 3 times on 3 separate TSA plates
  4. Using a pipette, spot 4 0.005 mL spots of chitinase enzyme on plate
    • 2 spots diluted to 10 mg/mL
    • 2 spots diluted 5 mg/mL
  5. Using a pipette, spot 6 0.005 mL spots of chitinase enzyme on plate
    • 2 spots diluted to 10 mg/mL
    • 2 spots diluted 1 mg/mL
    • 2 spots diluted 0.1 mg/mL
  6. Using a pipette, spot 4 0.005 mL spots of chitinase enzyme on plate
    • 2 spots diluted to 0.1 mg/mL
    • 2 spots diluted 0.01 mg/mL
  7. Use one plate without Yarrowia as a negative control
  8. Take one plate with Yarrowia spread and pipette 4 0.005 mL spots of PBS
    • Use plate as positive control
  9. Cover plates with a plastic bag and place all plates into an incubator
    • Incubate overnight at 27°Ce

Chitinase And Cinnamaldehyde Spotting Testing Against Yarrowia

  1. Pipette 0.300 mL of diluted Yarrowia culture on TSA plate
    • Use L-spreader to spread diluted Yarrowia culture
    • Let plates dry in fume hood for 30 minutes
  2. Make discs
    • Using a holepunch, create 100 discs
    • Wrap discs in aluminum foil
    • Autoclave discs
  3. Soak 25 discs in each of the following
    • Chitinase enzyme from Streptomyces Griseus diluted to 10 mg/mL in 1/32 diluted cinnamaldehyde
    • Chitinase enzyme from Streptomyces Griseus diluted to 5 mg/mL with 1/32 diluted cinnamaldehyde
    • Chitinase enzyme from Streptomyces Griseus diluted to 10 mg/mL with 1/32 diluted cinnamaldehyde
    • Chitinase enzyme from Streptomyces Griseus diluted to 5 mg/mL with 1/64 diluted cinnamaldehyde
  4. Place 4 of each of the previous discs on Yarrowia covered TSA plates
  5. Cover plates with a plastic bag and place plates in incubator
    • Incubate overnight at 27°C

Biofilm Assay Testing With Cinnamaldehyde

Growing a Biofilm

  1. Grow a culture of the wild-type Nissle overnight in a rich medium (i.e. LB)
  2. Dilute the overnight culture 1:100 into fresh medium for biofilm assays.
  3. Add 100 μL of the dilution per well in a 96 well dish.
  4. Add cinnamaldehyde at different volumes to create the desired concentrations
  5. Incubate the microtiter plate for 4-24 hrs at 37°C.

Staining the Biofilm

  1. After incubation, dump out cells by turning the plate over and shaking out the liquid.
  2. Gently submerge the plate in a small tub of water (i.e., use the bottoms of pipette tip boxes for P1000 pipetman as the tub). Shake out water. Repeat this process a two more times. This step helps remove unattached cells and media components that can be stained in the next step, and significantly lowers background staining.
  3. Add 125 μL of a 0.1% solution of crystal violet in water to each well of the microtiter plate. Wear gloves and a lab coat while making the solution. Use caution when weighing out the CV as the powder is hydroscopic and readily stains clothing, skin, etc.
  4. Incubate the microtiter plate at room temperature for 10-15 min.
  5. Rinse the plate 3-4 times with water by submerging in a tub of water as outlined above, shake out and blot vigorously on a stack of paper towels to rid the plate of all excess cells and dye.
  6. Turn the microtiter plate upside down and dry for a few hours or overnight.
  7. For qualitative assays, the wells can be photographed when dry.

Quantifying the Biofilm

  1. Add 125 μL of 30% acetic acid in water to each well of the microtiter plate to solubilize the CV.
  2. Incubate the microtiter plate at room temperature for 10-15 min.
  3. Transfer 125 μL of the solubilized CV to a new flat bottomed microtiter dish.
  4. Quantify absorbance in a plate reader at 550 nm using 30% acetic acid in water as the blank.

Procedure is a modified form of NCBI’s Microtiter Dish Biofilm Formation Assay (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182663/)

Contact us @iGEMLabPats or iGEM@carrollhs.org