Team:Harvard/Protocols

Harvard OPTI POLY

Protocols

Cloning

Heat Shock Transformation

Purpose

To visualize curli production in curli-producing colonies with fluorescence imaging

Materials
  • Competent Cells
  • plasmid DNA
  • 42ºC heat bath
  • Ice
  • Selection plates with appropriate antibiotic
Methods
  1. Thaw a tube of Competent E. coli cells on ice for 10 minutes.
  2. Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
  3. Place the mixture on ice for 30 minutes. Do not mix.
  4. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
  5. Place on ice for 5 minutes. Do not mix.
  6. Pipette 950 µl of room temperature SOC into the mixture.
  7. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
  8. Warm selection plates to 37°C.
  9. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
  10. Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.

*Procedure adapted from New England BioLabs' High Efficiency Transformation Protocol

Congo Red Agar Plates

Purpose

To visualize curli production in curli-producing colonies with fluorescence imaging

Materials
  • 10 g agar
  • 12.5 g LB
  • dH2O
  • Congo Red
Methods
  1. Add 10 g agar and 12.5 g LB to 500 mL of Milli-Q water
  2. Autoclaved for 30 minutes under “liquids” setting
  3. Put on bench until no longer scalding to the touch
  4. Remove the culture from the 25C incubator. Add 1 ml of the culture into a cleaned 2ml Eppendorf tube.
  5. Add Congo Red to a final concentration of 0.0025 g per 100 mL, arabinose to a final concentration of 100 μM, and kanamycin to a final concentration of 50 μg/mL
    • If using a different inducible promoter, adjust inducer accordingly
    • If using a different selection marker, adjust antibiotic accordingly
  6. Mix thoroughly
  7. Pour immediately into plates
  8. Pop superficial air bubbles with a Bunsen burner
  9. Let dry on bench for 2-3 hours with lids ajar

*Recipe adapted from Peter Nguyen et al. as detailed in “Programmable Biofilm-Based Materials from Engineered Curli Nanofibres” from Nature Communications

Picking Colonies with Fluorescence Imaging

Purpose

To pick colonies expressing highest levels of curli

Methods
  1. Take plates out of incubator
  2. Take fluorescent image of plate using a fluorescence imager, like FluorChem E
  3. Analyze image with a script for identifying the brightest colonies
  4. Using a colony picker, pick a single colony and place in a 5 mL falcon tube with liquid LB and appropriate antibiotic
  5. Place in incubator at 37ºC, 225 rpm overnight

*Script adapted from one created by Anton Kan in the Joshi Lab.

Miniprep

Purpose

To isolate DNA from cells for sequencing or other further experiments.

Materials
  • Buffer P1 (with RNase A added)
  • Buffer P2
  • Buffer N3
  • Buffer PB
  • Buffer EB (optional)
Methods
  1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.
  2. Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times.
  3. Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
  4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
  5. Centrifuge for 30–60 s. Discard the flow-through.
  6. Wash QIAprep 2.0 spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.
  7. Discard the flow-through, and centrifuge at full speed for an additional 1 min to remove residual wash buffer.
  8. Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min.

*Protocol from QIAprep Miniprep Handbook

Protein Expression Measurements

Congo Red Spin Down Assay

Materials
  • 1 mL LUDOX
  • H2O
  • 96 well plate
Methods
  1. Induce expression of curli by adding 1M IPTG to liquid culture, to a final concentration of 250 µM. For example, if you have 3 mL culture, add 0.75 µL of 1M IPTG solution.
  2. Incubate liquid culture overnight for optimal curli expression.
  3. Make 0.015% Congo Red solution by diluting 1% congo red solution. For example, 150 µl of 1% congo red solution in 10 ml DI water.
  4. Remove the culture from the 25C incubator. Add 1 ml of the culture into a cleaned 2ml Eppendorf tube.
  5. Pellet the cells by centrifuge at 6800 g (8000 rpm) at room temperature for 10 minutes.
  6. Remove the supernatant by decanting, and using a pipette to siphon off as much of the supernatant without disturbing the pellet.
  7. Resuspend cells in 1 mL of PBS gently by pipetting up and down with a pipette.
  8. Add 100 µl of 0.015% congo red solution to the tube. Mix gently by pipetting. For the control tube, add the congo red solution to 1 ml of pure PBS.
  9. Leave the solution at room temperature for 10 minutes
  10. Pellet by centrifuging at 14,000 rpm at room temperature for 10 minutes.
  11. Add 150 µl of the supernatant to a well of 96-well plate along with pure PBS, and control PBS-Congo Red.
  12. Using a plate reader, measure absorbance at wavelength 490 nm.

*Protocol courtesy of Bom Praveschotinunt of Wyss Institute, Joshi Lab

Filtration

Purpose

To visualize curli production in curli-producing colonies with fluorescence imaging

Materials
  • 500 mL cell culture
  • Guanidinium Chloride (GdmCl)
  • 47 mm polycarbonate membrane with 10 um pores
  • dH2O
  • 2uM MgCl2
  • 1.5 U/mL of Benzonase, SigmaAldrich
  • 5% aqueous SDS
Methods
  1. Prepare an overnight 500 mL culture of cells with appropriate selection marker and the appropriate inducing agent
  2. Add guanidinium chloride (GdmCl) to a final concentration of 0.8 M
  3. Let sit for 1-2 hours at 4C
  4. Remove the culture from the 25C incubator. Add 1 ml of the culture into a cleaned 2ml Eppendorf tube.
  5. Vacuum filter cell media on a 47 mm polycarbonate membrane with 10 μm pores until the filter is clogged
  6. Incubate the filtered biomass with 5 mL of 8 M GdmCl for 5 minutes followed by vacuum filtration and 3 subsequent washes with DI water
  7. Incubate the remaining filtered biomass with 5 mL of an aqueous solution with 2 μM MgCl2 and 1.5 U/mL of Benzonase®, SigmaAldrich for 10 minutes
  8. Pop superficial air bubbles with a Bunsen burner
  9. Vacuum filter the liquid followed by 3 subsequent washes with DI water
  10. Incubate in 5 mL of 5% aqueous SDS (m/v) for 5 minutes
  11. Vacuum filter the liquid followed by 3 subsequent washes with DI water
  12. Scrape biomass from filter with a spatula

*Procedure adapted from Neel Joshi et al. as detailed in “Scalable Production of Genetically Engineered Nanofibrous Macroscopic Materials via Filtration” from ACS Biomaterials Science & Engineering