Team:Pittsburgh/Experiments

 






Protocols

 


Buffers & Solutions

Antibiotic Concentrations

Antibiotic Stock Concentration (mg/mL) Antibiotics vol for 20 mL LB plates (uL) Antibiotics vol for 1 mL LB broth
Kan 30 33.33 1
Chlor 30 20 0.67
Strep 50 12 0.60
Amp 100 20 0.50

1x SDS Running Buffer (1L)

  1. 14.4 glycine
  2. 3.0g Tris Base
  3. 1g SDS
  4. (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

5X KCM; Sterile filter, store at 4°C

Final conc. Stock conc. Volume (ml)
0.5 M KCl 2.5 M 6
0.15 M CaCl2 1 M 2.5
0.25 M MgCl2 1 M 7.5
ddH2O to 30 mL total 25

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

Transformation Storage Buffer (TSB); Sterile filter and store at 4°C

Final conc. Stock conc. Volume (ml)
LB broth, pH 6.5 2x 20.5
10% PEG 3350 50% 10
5% DMSO 100% 2.5
10 mM MgCL2 1 M 0.5
10 mM MgSO4 1 M 0.5
ddH2O to /td> 50 mL total 15 mL


Competent Cells

Preparation of Competent Cells

  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

Preparation of Competent Cells

PCR (Phusion, Q5 2x master mix)

PCR Component 1 5 9 11
5X GC Buffer 10 50 90 110
dNTPs (10mM) 1 5 9 11
PCR Grade Water 20 100 180 220
DMSO 1.5 7.5 13.5 16.5
Betaine (5 M) 13 65 117 143
Sense Primer (10 μM) 1.5 7.5 13.5 16.5
Antisense Primer (10 μM) 1.5 7.5 13.5 16.5
Template DNA 1 5 9 11
Phusion DNA Polymerase (1 U/μL) 0.5 2.5 4.5 5.5
Total Volume 50 250 450 550
PCR Component 1 5 9 16
2X Master Mix (DreamTaq) 5 25 45 80
Forward Primer (10 μM) 0.5 2.5 4.5 8
Reverse Primer (10 μM) 0.5 2.5 4.5 8
Betaine (5 M) 2.6.5 13 23.4 41.6
DMSO 0.5 2.5 4.5 8
Water 0.9 4.5 8.1 14.4
Total Volume 10 50 90 160
Step Temperature Time
Initial Denaturation 98°C 30 seconds
25 cycles 98°C
45-72°C
72°C 		5-10 seconds
10- 30 seconds
60- 120 seconds per kb
Final Extension 72°C 5-10 minutes
Hold 4°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

  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 supernatent
  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

Plasmid Screening (Colony PCR)

PCR Component 1 5 9 16
2X Master Mix (DreamTaq) 5 25 45 80
Forward Primer (10 μM) 0.5 2.5 4.5 8
Reverse Primer (10 μM) 0.5 2.5 4.5 8
Betaine (5 M) 2.6.5 13 23.4 41.6
DMSO 0.5 2.5 4.5 8
Water 0.9 4.5 8.1 14.4
Total Volume 10 50 90 160
Step Temperature Time
Initial Denaturation 98°C 30 seconds
25 cycles 98°C
45-72°C
72°C 		5-10 seconds
10- 30 seconds
60- 120 seconds per kb
Final Extension 72°C 5-10 minutes
Hold 4°C

Preparation for Sequencing

  • Set up sterile field
  • Prepare 2 mL of LB per sample that you want to sequence; add 20 μL of culture to LB and add 2 μL of specified antibiotics
  • Dilute to OD 0.4

Microscope preparation (basic)

  1. Add 10uL of sterile water to a pcr tube
  2. Use a pipet-tip to transfer some cells from plate to the water by lightly scraping cells off the plate and swirling it in the water
  3. Plate 2 μL of cells onto a microscope slide, place a slide cover over sample

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

Protein Expression- 1L culture

Expression Optimization Protocol

  1. Inoculate 50 mL of [antibiotic]+ LB-Miller overnight at 37°C in a 150 mL flask
    • 10 mL of overnight culture per L of large culture
    • Flask 3x larger than media volume
    • ~5PM to 9AM
  2. Inoculate large (1L) culture w/10 mL overnight culture
    • 1L with antibiotic (50 μg/mL AMP)
    • Begin at 9AM
  3. Grow to OD600 =0.5
    • Can use nanodrop or plate reader to measure
    • Blank with LB
    • ~noon
  4. Induce expression with 1mM [IPTG]final
    • [IPTG]final = 1 mL 1M IPTG/L culture
    • Aliquot into 1mL in 1.7 mL Eppendorf tubes
  5. Grow at 37°C for 3-4 hours
    • 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
    • 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
    • Pour off liquid into sink (possibly with bleach)
    • Repeat for all of the same culture into the same bottle
  10. For long term storage, resuspend in resuspension buffer.
    • 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

Gel staining

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)
    • 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
  3. Destain the gel by rinsing three times and then soaking in deionized water
    • Replace solvent several times during the destaining process or add a kimwipe to absorb residual Coomassie Blue.
  4. Leave

Temperature Sensitive Dimers

Induction Protocol for Heat Sensitive Dimers (Piraner)

  1. Pick a colony and grow overnight at 30C in 2xYT medium (what the heck is that? Gonna use LB)
  2. In 20 hours (could also be adjusted to 14-16 hours), dilute to OD600 = 0.1 (approx. 30uL into 1 mL LB)
    • Use the dilution instead of measuring the OD
    • Place diluted culture back in 30 Celsius
  3. After another 1 hour 15-30 min start checking culture OD600s
    • When ODs are 0.5-0.6, begin aliquoting
    • NOTE: different constructs grow at different rates, which can be tricky to coordinate; if there is an overshoot with the OD, you can re-dilute in more LB and try again
  4. Aliquot 25uL E.coli into PCR tubes/strips (to give bacteria as much air as possible)
    • When moving culture to plate reader for OD measurement later, add 75uL LB, mix, and transfer 90uL into a 96 well plate
  6. Incubate in the Thermocycler either at:
    • A set temperature
    • Gradient for 12 hours (this was the standard experimental time, but we can start seeing induction earlier)
  7. Gradient for 12 hours (this was the standard experimental time, but we can start seeing induction earlier)

Initial testing for GFP expression

  1. Dilute to OD600 = 0.1 in LB (Sigma) with 100 μg/mL ampicillin for each culture and propagate to 30, 250 rpm for 1.5h. At OD600, dispense culture in 25 μL aliquots in PCR tubes.
    3. Set up 3 PCR blocks (37°C, 42°C, 45°C respectively).

Binding MNPs to E.coli surface

Amine-functionalized MNPs

  1. Thaw 1 tube of competent cells on ice. Add 200 uL of LB.
  2. Spin down. Pipette out the supernatent. Add about 500 uL of PBS (10 mH, pH 7.0).
  3. Check OD.
  4. Add 50 uL AF-MNPs to the suspension for a final concentration of 0.5 mg mL-1.
  5. Incubate for about 15 min at 250 rpm.
  6. To measure how much of bacteria has been captured by the MNPs, use an external magnet to separate out the bacteria. Then, measure the OD600 of the supernatant.
  7. After magnetic separation, resuspend the nanoparticle-bacteria conjugate in 100 uL of PBS.
  8. Add 0.5 uL onto mesh carbon-coated copper grids.
  9. After drying, the sample can be subjected to TEM analysis.

Chitosan-functionalized MNPs

  1. Dissolve 20 mg of chitosan to 1 M acetic acid with a final volume of 100 mL.
  2. Add 70 mg of SPION to the solution.
  3. Allow the solution to stir for 18 h.
  4. This resulting solution should be a homogenous brown solution.
  5. CS-MNPs can then be collected by magnetic separation and rinsed with water and ethanol to remove free chitosan.
  6. Resulting CS-MNPs can then be dried at 60 °C under vacuum overnight.
  7. Grow E.coli in LB media to log phase (OD 600 nm ~ 0.6).
  8. Centrifuge the bacteria at 8000 rpm for 3 min and wash the cells several times with PBS.
  9. Serially dilute the cells in PBS to obtain 105 CFU mL-1 suspensions.
  10. Treat the cells with 200 μg mL-1 CS-MNPs for 6 h.
  11. Centrifuge the mixture at 8000 rpm for 3 min and rinse it twice with PBS

Biotinylated MNPs to Streptavidin on E.coli

  1. Verify that streptavidin is expressed at the surface using biotinylated GFP.
  2. Use E.coli that has already been transformed with streptavidin plasmid.
  3. Dissolve biotinylated MNPs in solution.
  4. Add the MNPs to suspension of E.coli.