Team:Harvard/Protocols
Protocols
Cloning
Purpose
To visualize curli production in curli-producing colonies with fluorescence imaging
Materials
- 10 g agar
- 12.5 g LB
- dH2O
- Congo Red
Methods
- Add 10 g agar and 12.5 g LB to 500 mL of Milli-Q water
- Autoclaved for 30 minutes under “liquids” setting
- Put on bench until no longer scalding to the touch
- Remove the culture from the 25C incubator. Add 1 ml of the culture into a cleaned 2ml Eppendorf tube.
- 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
- Mix thoroughly
- Pour immediately into plates
- Pop superficial air bubbles with a Bunsen burner
- 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
Protein Expression Measurements
Materials
- 1 mL LUDOX
- H2O
- 96 well plate
Methods
- 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.
- Incubate liquid culture overnight for optimal curli expression.
- 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.
- Remove the culture from the 25C incubator. Add 1 ml of the culture into a cleaned 2ml Eppendorf tube.
- Pellet the cells by centrifuge at 6800 g (8000 rpm) at room temperature for 10 minutes.
- Remove the supernatant by decanting, and using a pipette to siphon off as much of the supernatant without disturbing the pellet.
- Resuspend cells in 1 mL of PBS gently by pipetting up and down with a pipette.
- 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.
- Leave the solution at room temperature for 10 minutes
- Pellet by centrifuging at 14,000 rpm at room temperature for 10 minutes.
- Add 150 µl of the supernatant to a well of 96-well plate along with pure PBS, and control PBS-Congo Red.
- Using a plate reader, measure absorbance at wavelength 490 nm.
Protocol Courtesy of Bom Praveschotinunt of Wyss Institute, Joshi Lab
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
- Prepare an overnight 500 mL culture of cells with appropriate selection marker and the appropriate inducing agent
- Add guanidinium chloride (GdmCl) to a final concentration of 0.8 M
- Let sit for 1-2 hours at 4C
- Remove the culture from the 25C incubator. Add 1 ml of the culture into a cleaned 2ml Eppendorf tube.
- Vacuum filter cell media on a 47 mm polycarbonate membrane with 10 μm pores until the filter is clogged
- 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
- 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
- Pop superficial air bubbles with a Bunsen burner
- Vacuum filter the liquid followed by 3 subsequent washes with DI water
- Incubate in 5 mL of 5% aqueous SDS (m/v) for 5 minutes
- Vacuum filter the liquid followed by 3 subsequent washes with DI water
- 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
