Team:Pittsburgh/Experiments


 




Experiments

 

Antibiotic Concentration



SDS Page Gel

    1x SDS Running Buffer (1L)
      14.4 glycine
      3.0g Tris Base
      1g SDS
      (fill to 1L with millipore dH2O)


    Coomassie Stain for Protein Gels (1L), store in dark
      60-80 mg Coomassie G-250
      1 L MilliQ water; Stir for several hours to dissolve


    SDS Sample Buffer (3x); Store at -20°C
      1.5 mg Bromophenol Blue (BPB)
      0.60 g SDS
      3.0 mL GLycerol
      3.9 mL 0.5 M Tris-HCl
      1.5 mL β-mercaptoethanol
      __ mL dH2O to make final volume of 10 mL


    Expression Optimization Protocol
  1. Inoculate 50 mL of [antibiotic]+ LB-Miller overnight at 37°C in a 150 mL flask
    1. 10 mL of overnight culture per L of large culture
    2. Flask 3x larger than media volume
    3. Approximately 5PM to 9AM
  2. Inoculate large (1L) culture w/10 mL overnight culture
    1. 11L with antibiotics (50 μg/mL AMP)
    2. Begin at 9AM
  3. Grow to OD600=0.5
    1. Can use nanodrop or plate reader to measure
    2. Blank with LB
    3. Approximately Noon
  4. Induce expression with 1mM [IPTG]final
    1. [IPTG]final = 1 mL 1M IPTG/L culture
    2. [IPTG]final = 1 mL 1M IPTG/L culture
  5. Grow at 37°C for 3-4 hours
    1. Grow at 37°C for 3-4 hours
    2. Finish around 4PM
  6. Take sample (1 mL) of cells, pellet at 21,000 xg for 1 minute
  7. Resuspend pellet (1 mL sample) in 50 μL of denaturing sample buffer. Heat at 75-100°C for 5-10 minutes to lyse cells.
  8. Shear DNA by vortexing and pellet again at 21,000 xg for 5 minutes before loading on a gel to test expression
    1. Most likely 20% or 17% gel, 4-20% works as well
  9. Run gel and spin 1 L cultures down in Nalgene 1 L bottles at 4000 xg, 10-15 minutes
    1. Pour off liquid into sink (possibly with bleach)
    2. Repeat for all of the same culture into the same bottle
  10. For long term storage, resuspend in a resuspension buffer.
    1. 50 mM HEPES-KOH pH8.0, 500 mM NaCl
  11. Pellet resuspended cells into 50 mL tubes (conical) at 4000 xg, 10 minutes
  12. Pour off liquid and freeze at -80°C until ready for purification


    PAGE + Coomassie Blue
  1. Rinse the gel in deionized water three times to remove any residual SDS
  2. Soak the gel in enough Coomassie Blue to cover the surface and let sit for 10 minutes, shaking occasionally (or use a shaker table)
  3. When you get tired of waiting for the gel to stain, place it in the microwave for 15 seconds at a time, being careful not to overheat the gel
  4. Destain the gel by rinsing three times and then soaking in deionized water
    5. Replace solvent several times during the destaining process or add a kimwipe to absorb residual Coomassie Blue


Preparation of Competent Cells



    Protocol
  1. Plate cells on LB plate. Pick an isolated colony and grow cells in 5 mL LB medium overnight
  2. Transfer 1 mL culture to 100 mL LB media and grow at 37°C to OD600=0.4-0.6 in the morning; takes around 3 hrs
  3. Place culture on ice for 5 min
  4. Pellet cells at 4000 x g for 10 minutes
  5. Resuspend cells in 1/10 volume of chilled TSB
  6. Incubate on ice for 5 minutes
  7. Subpackage into tubes (100 μL aliquots) and freeze in liquid nitrogen
  8. Store at -80°C

DNA Gel Electrophoresis



    Preparing Gel
  1. Weigh 1g of agarose to 100 mL of 1x TAE buffer solution
  2. Microwave and stir until fully dissolved (about 2 minutes)
  3. When almost cooled, add 2 μL of ethidium bromide (careful)
  4. Place combs, desired side down, and pour solution
  5. For 50uL PCR product, use thicker side of comb


    Running Gel
  1. Pour 1x TAE buffer till entire gel is covered
  2. Load sample and ladder
  3. Plug in the electrodes in a Negative to Positive direction
  4. Run for 25 minutes at 125V
  5. Turn off electrodes before removing cover
  6. Look at gel under UV
  7. Use a razor cut and transfer band of DNA to a clean eppendorf tube
  8. Repeat for each column
  9. Freeze at -20°C or continue onto gel purification protocol
  10. Dispose of gel into specified ethidium bromide waste

DNA Plasmid Experiments



    MiniPrep
  1. Transfer 10 mL of cell culture, 1.5 mL aliquots at a time into 1 Eppendorf tube, using 1 tube for each culture.
  2. Centrifuge for 1 min to pellet cells; discard the supernatant
  3. Add 250uL of resuspension buffer (or water) and resuspend
  4. Pipet up and down to fully resuspend cells
  5. Add 350uL of Lysis solution
  6. Invert 4-6 times
  7. Add 350uL of neutralization solution
  8. Invert 4-6 times
  9. Centrifuge for 5 minutes to pellet cell walls and chromosomal DNA
  10. Transfer supernatant to filter (blue)
  11. Centrifuge for 1 minute
  12. Discard flow through
  13. Wash twice with wash buffer
  14. Collect with sterile water


    Transformation
  1. Combine 20 μL 5x KCM + x μL DNA (~ 10-100 ng) + ddH2O to 100 μL
  2. Add 100 μL competent cells, mix gently and incubate on ice for 20 minutes
  3. Heat shock at 42°C for 90 sec
  4. Place on ice for 1 minute
  5. Add 400-600 μL LB to cells
  6. Shake for 45 min to 1 hour at 37°C
  7. Plate 50-250 μL cell

PCR



Cycling Steps



    H2O2 Gibson Assembly
  1. Prepare the Gibson Assembly Master Mix as noted below protocol
    1. Store at -20°C in 15 μL aliquots
  2. Thaw a 15 μL aliquot of the Gibson assembly master mix and keep on ice until use
  3. Measure the DNA concentration (ng/μL) of each assembly piece
  4. Add 100 ng of linearized vector backbone and equimolar amounts of other assembly pieces to the thawed 15 μL master mix (see table below)
  5. Incubate the assembly reaction at 50°C for 60 minutes, and then place on Ice
  6. Transform 5 μL of the assembly reaction into 100 μL of competent DH5α E. coli







FimH Binding Experiments



    IPTG Induction
  1. Inoculate 2 seperate falcon tubes containing 2 mL of LB broth and 50 ug/mL of kanamycin with BL21 bearing histidine and his/SNAP combination plasmids.
  2. Incubate the culture overnight at 37°C while shaking at 250 rpm.
  3. Perform a 1:100 dilution of the overnight culture with 50 ug/mL of kanamycin and incubate the culture 3-4 hrs at 37°C while shaking at 250 rpm.
  4. Read the absorbance of the culture via OD at 600 nm (it should be around 0.5-0.6).
  5. Add IPTG to obtain a final concentration of 0.5 mM IPTG.
  6. Incubate for 3-4 more hours at 37°C while shaking at 250 rpm.


    Nickel Bead Assay
  1. Inoculate 2 seperate falcon tubes containing 2 mL of LB broth and 50 ug/mL of kanamycin with BL21 bearing histidine and his/SNAP combination plasmids.
  2. Prepare an additional falcon tube with 2 mL of LB broth and inoculate it with untransformed BL21 containing wild type fimH.
  3. Incubate the cultures overnight at 37°C while shaking at 250 rpm.
  4. Dilute all three cultures by a factor of 100 (1:100 dilution) in 2 mL of LB broth.
  5. Add IPTG to all three cultures such that the final concentration of IPTG is 0.5 mM.
  6. Add kanamycin stock (30 mg/mL) to the cultures with E.Coli cells containing the histidine tag and SNAP tag and histidine tag combination.
  7. Grow the cultures for 2-4 hours until the O.D of the culture has reached 0.5-0.6. r.
  8. Centrifuge the culture at 1400 rpm for 2 minutes and resuspend in 5 mL of PBS. Repeat 2-3 times to wash the bacteria.
  9. Add 100 uL of the cultures to 3 separate microscope slides along with 40 uL of Ni-NTA beads.
  10. View each slide under a microscope


    Yeast Agglutination Assay
  1. Inoculate 2 seperate falcon tubes containing 2 mL of LB broth and 50 ug/mL of kanamycin with BL21 bearing histidine and his/SNAP combination plasmids.
  2. Prepare an additional falcon tube with 2 mL of LB broth and innoculate it with untransformed BL21 containing wild type fimH.
  3. Incubate the cultures overnight at 37°C while shaking at 250 rpm.
  4. Pellet the cells by centrifuging at 14,000 rpm for 2 min. Completely remove the supernatant and resuspend the cells in PBS.
  5. Prepare a glass microscope slide for each culture. Add 25 uL of the resuspended bacteria to each culture and 25 uL of Saccharomyces cerevisiae (yeast) suspended in PBS.
  6. Start a timer and note the time at which agglutination begins.


    Imaging Protocol for Histidine and Histidine tag combination plasmids
  1. Inoculate 2 seperate falcon tubes containing 2 mL of LB broth and 50 ug/mL of kanamycin with BL21 bearing histidine and his/SNAP combination plasmids.
  2. Inoculate a seperate falcon tube containing 2 mL of LB broth with BL21 cells containing native fim-H.
  3. Incubate the culture overnight at 37°C while shaking at 250 rpm..


OxyR Experimentation and Plasmid Design



    Designing Plasmids with OxyR Binding Sites
  1. Delete the original promoter, operator, and ribosomal binding site (RBS).
  2. Add in the different OxyR binding sites, which should be upstream of genes regulated by OxyR, including katG, dps, and grxA.


    H2O2 Growth Assay
  1. Grow cells overnight. Inoculate the culture into 1.5 mL tubes and dilute it 1:50.
  2. Add different concentrations of H2O2 to the tubes.
  3. Measure OD600 with a plate reader.


    H2O2 Induction
  1. Grow 2mL of cultures of the OxyR mutants and pUC-19 positive control overnight in 15 mL falcon tubes.
  2. Dilute concentrate to 1:100
  3. Create dilutions for H2O2
    1. 1 mM
    2. 500 μM
    3. 250 μM
    4. 100 μM
    5. 500 μM
  4. Incubate the diluted cultures at 37°C, shaking at 250 RPM, taking a sample of each every 30 minutes for 3 hours (0.2 mL H2O2:1.8 mL culture).
  5. Pellet out the cells in the remaining liquid culture.
  6. Freeze at -80°C if not running lacZ assay the same day.
  7. Resuspend cells in lysis buffer and sonicate.
  8. Lyse cells and run lacZ assay


    LacZ Assay
  1. Prepare lysis buffer.
    1. 60 mM Na2HPO4
    2. 40 mM NaHPO4
    3. 10 mM KCl
    4. 1 mM MgSO4
    5. 36 mM β-mercaptoethano
    6. 166 μl/ml 7 lysozyme
    7. 1.1 mg/ml ONPG
    8. 6.7% PopCulture reagen
  2. Add 120 μL of culture with 80 μL of lysis buffer.
  3. Read absorbance at OD420.