Team:Toronto/Project/Elementss

STIMULI PROTOCOLS

Prelude
We looked at the effect of various stimuli on the polysaccharides, proteins, and structures involved in the various stages of a biofilm. In particular, we looked at varying concentrations of nutrients, physical environments, and media.
For nutrients, we chose three levels of stimulation – zero stimulation, medium stimulation, and maximum stimulation. Their effects on the biofilm response were then observed and analysed through a battery of assays looking at the levels of biofilm components.
The stimuli were administered and the cell cultures were incubated for 48 (±3) hours at 23 degrees Celsius, which is the optimum temperature for biofilm growth (White-Ziegler et al. 2008). As bacteria turn their environment acidic as a result of anaerobic metabolism, buffered LB was used using the phosphate salts, potassium phosphate monobasic and potassium phosphate dibasic. pH 7 was adjusted for using 0.1 M concentration in Luria Broth. The E. coli cells were incubated in buffered LB to maintain pH 7 and eliminate the extraneous variable of acidity. The stock solutions for the stimuli were also created in buffered LB to prevent uneven nutrient levels; the phosphate salts are a systematic error across all inoculations and can thus be ignored.
In continuation with the project, we are planning to optimize results by combining the treatments which give the best results.

Ethanol
Reasoning:
Given the large quantity of literature on the effect of ethanol on biofilm, it is a very important stimulus. The ethanol concentrations being tested in this experiment are 0.53% and 2%. Cegelskiet al. (2012) found that the level of some proteins involved in biofilm formation increases in the presence of ethanol. They also found UT189 strain colonies to be 55% larger (colony morphology assay). Cell viability is compromised at concentrations exceeding 4%.
Materials for Ethanol stimulus:
• Overnight cultures of E. coli
•40% EtOH solution prepared in phosphate buffered LB
• 96-well plate

Protocol:
1. Grow overnight culture of E. coli in phosphate buffered media.
2. Add 200 µL of 1:100 dilution of overnight culture along with 100 µL of 1.6% EtOH in buffered LB solution into a well in a 96-well plate to obtain a final EtOH concentration of 1%.
3. Add 200 µL of 1:100 dilution of overnight culture along with 100 µL of 6% EtOH in buffered LB solution into a well in a 96-well plate to obtain a final concentration of 2%.
4. Incubate in darkness at 23 degrees C for 48 hours.
Lim et al. (2012) Dimethyl sulfoxide and ethanol elicit increased amyloid biogenesis and amyloid-integrated biofilm formation in Escherichia coli. Appl Environ Microbiol 78:3369-78.

Carbon Source

Sucrose
Reasoning:
The concentrations of sucrose being tested are 0.5 M and 0.1 M. Hagiwara et al. (2009) found the growth curves to show optimum biofilm at a 0.1 M concentration. E. coli biofilm formation decreases at high osmolarity - sucrose is being used here to test osmolarity as a non-ionic solute.
Materials for sucrose stimulus:
• Overnight cultures of E. coli
• 2 M sucrose stock solution prepared in phosphate buffered LB
• 96-well plate
Protocol:
1. Grow overnight cultures in phosphate buffered media.
2. Add 200 µL of 1:100 dilution of overnight culture along with 100 µL of
0.3% sucrose solution made in buffered LB solution into a well in a 96-well plate to obtain a final concentration of 0.1 M.
3. Add 200 µL of 1:100 dilution of overnight culture along with 100 µL of 1.5% sucrose in buffered LB solution into a well in a 96-well plate to obtain a final concentration of 0.5 M.
4. Incubate in darkness at 23 degrees C for 48 hours.
Kawarai et al. (2009) Biofilm formation by Escherichia coli in hypertonic sucrose media. J Biosci Bioeng 107:630-5.

Indole
Reasoning:
Indole has an overall negative effect on biofilm. The concentrations being tested are 500 micromolar and 300 micromolar.
Materials:
• 0.0009 M Indole in phosphate buffered LB
• Overnight culture diluted in a 1:100 ratio
Protocol:
1. 300 µM
  a. 1:100 dilution of overnight culture was inoculated in buffered LB.
  b. 200 µl of the dilution was added to the well.
  c. 100 µl of 0.0009 M indole was added to the well.
  d. The culture was incubated for 48 hours at 23 Celsius.
2. 500 µM
  a. 1:100 dilution of overnight culture was incubated in buffered LB.
  b. 200 µl of the dilution was added to the well.
  c. 100 µl of 0.0015 M was added to the well.
  d. The culture was incubated for 48 hours at 23 Celsius.
Bansal et al. (2007) Differential effects of epinephrine, norepinephrine, and indole on Escherichia coli O157:H7 chemotaxis, colonization, and gene expression. Infect Immun 75:4597-607.

DMSO
Reasoning:
Effects are similar to that of ethanol. The levels of certain proteins that regulate biofilm formation were 3-3.9 times higher than baseline, and there was an increase in curli (Lim et al., 2012). The concentrations being tested are 4% and 2%.
Materials list:
• Dimethyl sulfoxide in phosphate buffered LB
• Overnight culture diluted in a 1:100 ratio
Protocol:
1. 2%
  a. 1:100 dilution of overnight culture was incubated in buffered LB.
  b. 200 µl of the dilution was added to the well.
  c. 100 µl of 6% DMSO was added to the well.
  d. The culture was incubated for 48 hours at 23 Celsius.
2. 4%
  a. 1:100 dilution of overnight culture was incubated in buffered LB.
  b. 200 µl of the dilution was added to the well.
  c. 100 µl of 12% DMSO was added to the well.
  d. The culture was incubated for 48 hours at 23 degrees Celsius
Lim et al. (2012) Dimethyl sulfoxide and ethanol elicit increased amyloid biogenesis and amyloid-integrated biofilm formation in Escherichia coli. Appl Environ Microbiol 78:3369-78.

Media

NaCl stimulus
Reasoning:
The concentrations being tested are 0.3 M and 0.5 M. NaCl is being tested for its effect as an osmolite in medium. Park et al. (2012) found NaCl-free to increase attachment and the capacity of biofilm formation under salt (0.1-0.3 M) was similar to that of the control. Zogaj et al. (2001) says that the bcs genes were expressed under 0.5 M - it is unclear whether that is similar to the levels found in the wild type strain.
Materials:
• Phosphate buffered LB without NaCl
• Overnight culture diluted in a 1:100 ratio
Protocol:
1. Sodium chloride free Luria Broth
  a. 275 µl of NaCl-free LB was added to the well.
  b. 25 µl of overnight culture was added to the well.
  c. The culture was incubated for 48 hours at 23 degrees Celsius.
Yeom et al. (2012) Effects of non-ionic solute stresses on biofilm formation and lipopolysaccharide production in Escherichia coli O157:H7. Res Microbiol 163:258-67

Terrific Broth (TB)
Reasoning:
Verma et al. (2010) found the least amount of biofilm to be produced at 37 degrees in TB.
Materials:
• 1:100 dilution of overnight culture grown in phosphate buffered media
Protocol:
1. Prepare Terrific Broth media by autoclaving a 900 mL solution of double distilled water containing 12 g tryptone, 24 g of yeast extract and 4 mL of glycerol.
2. Add 100 mL of a sterile solution containing a final concentration of 0.17 M KH²PO⁴, 0.72 M K²HPO⁴.
3. Add 275 µl of Terrific Broth to the well.
5. Add 25 µl of overnight culture to the well.
6. Incubate the culture for 48 hours at 23 degrees.
Prüss et al. (2010) Environmental and genetic factors that contribute to Escherichia coli K-12 biofilm formation. Arch Microbiol 192:715-28.

Tryptic Soy Broth
Reasoning:
Verma et al. (2010) found most biofilm to be produced at 37 degrees in TSB media.
Materials:
• TSB media
• Overnight culture
Protocol:
1. To 900 mL of double distilled water, add 17 g Tryptone, 3 g peptone-B, 2.5 g glucose, 5 g NaCl and 2.5 g of K2HPO4 to prepare TSB media.
2. 275 µl of Tryptic Soy Broth was added to the well.
3. 25 µl of overnight culture was added to the well.
4. The culture was incubated for 48 hours at 23 degrees.

Phosphate buffered Luria Broth
Reasoning:
To prevent fluctuations in pH as cell growth progresses, phosphate buffered Luria Broth media is used in place of regular LB.
Materials:
• Phosphate buffered LB
• overnight culture
Protocol:
1. 275 µl of buffered Luria Broth was added to the well.
2. 25 µl of overnight culture was added to the well.
3. The culture was incubated for 48 hours at 23 degrees.

Unbuffered Luria Broth
Reasoning:
The regular recipe for LB was used and this was used to establish a baseline to compare all other media.
Materials:
• LB
  o 10 g NaCl/L ^ddH²O
  o 5 g yeast extract/L ^ddH²O
  o 10 Tryptone/L ^ddH²O
• Overnight culture
Protocol:
1. 275 µl of unbuffered Luria Broth was added to the well.
2. 25 µl of overnight culture was added to the well.
3. The culture was incubated for 48 hours at 23 degrees

Acidic Luria Broth (0.5 M potassium phosphate monobasic)
Reasoning:
Acidic conditions are one set of stimuli that increase levels of a protein that normally increases motility and decreases the biofilm response.
Materials:
• Acidic buffered LB
  o 0.5 M Potassium Phosphate Monobasic
• Overnight culture
Protocol:
1. 275 µl of acidic LB was added to the well.
2. 25 µl of overnight culture was added to the well.
3. The culture was incubated for 48 hours at 23 degrees.

INOCULATION SERIES

In order to complete our stimuli and assays most efficiently we combined our protocols into a master protocol. 2 strains are assayed on 3 plates; the well organization scheme is such that fully characterizing one strain requires exactly 1.5 plates. Our plate organization and pipetting schemes are described in the following pdfs. found
HERE (Strain 1, Plates 1 and 2) and HERE (Strain 2, Plates 2 and 3).