Team:Toronto/Project/Elements

From 2013.igem.org

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(colony morphology assay). Cell viability is compromised at concentrations exceeding 4%.</br>
(colony morphology assay). Cell viability is compromised at concentrations exceeding 4%.</br>
Materials for Ethanol stimulus:</br>
Materials for Ethanol stimulus:</br>
-
** Overnight cultures of E. coli<br/>
+
Overnight cultures of E. coli<br/>
-
**40% EtOH solution prepared in phosphate buffered LB<br/>
+
•40% EtOH solution prepared in phosphate buffered LB<br/>
-
** 96-well plate<br/>
+
96-well plate<br/>
<p style = "font-size:18px;"><b>Protocol:</b><br/>
<p style = "font-size:18px;"><b>Protocol:</b><br/>
1. Grow overnight culture of E. coli in phosphate buffered media.<br/>
1. Grow overnight culture of E. coli in phosphate buffered media.<br/>
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Escherichia coli O157:H7 chemotaxis, colonization, and gene expression.<i> Infect  
Escherichia coli O157:H7 chemotaxis, colonization, and gene expression.<i> Infect  
Immun</i> <b>75</b>:4597-607.
Immun</i> <b>75</b>:4597-607.
 +
 +
</p>
</p>

Revision as of 00:29, 28 September 2013

STRAINS TESTED


BW25113 WT
MG1655 WT BW25113 pEBS-csgD
BW25113 pEBS-csgD
The protein CsgD is a regulator of genes involved in curli assembly, induced in mid-exponential phase that are active in stationary phase. It positively controls σSexpression; its deletion should in principle interfere with biofilm formation. [1]
BW25113 pEBS-fimB
The gene fimB mediates off to on switching of fim operon, while fimE mediates on to off switching of the fim operon. Its deletion should in principle repress the fim operon, while its overexpression should in principle encourage fimbriae formation. [2]
BW25113 pEBS-mlrA
MlrA stands for a merR-like regulator A, which regulates curli production. Its overexpression should in principle suppress curli formation, while its deletion should in principle encourage curli formation. [3]
BW25113 pEBS-ompA
The protein OmpA influences cellulose production by repressing cellulose production with CpxRA stress response system; it is overexpressed in biofilm formation and repressed when cells are exposed to visible light. [4]
BW25113 pEBS-ydeH
YdeH is a diguanylate cyclase, where its product regulates biofilm formation and motility. Its deletion has been shown to reduce surface attachment of the cell, its overexpression has been shown to reduce motility as well as flagella. [5]
BW25113 ΔfimA
FimA is the major subunit of E. coli type 1 (mannose sensitive) fimbriae, also known as pili. Pili are made of ~1000 units of FimA. Its deletion would remove the ability of a cell to produce fimbriae.[6]
BW25113 ΔompA
The protein OmpA influences cellulose production by repressing cellulose production with CpxRA stress response system; it is overexpressed in biofilm formation and repressed when cells are exposed to visible light.[4]
BW25113 ΔdosC
A heme-containing, oxygen-sensitive diguanylate cyclase. Its overexpression drives the cell to tend towards stationary phase physiology, leading to more biofilm and less motility. DosC expression is dependent on σS .[7]
BW25113 ΔompX
Adhesion of the cell on a surface represses ompX. Its deletion leads to increase in cell-surface contact in fimbriated E. coli, the opposite occurs in non-fimbriated E. coli.[8]
BW25113 ΔpgaB
PgaB is involved in the transport of PGA (an extra-membrane polysaccharide) across the outer membrane), which is involved in biofilm formation. Strains with mutant pgaB form less biofilm. Its expression is increased in environments with 1% ethanol or NaCl. [9]
BW25113 ΔrcsA
+ regulator of capsular polysaccharide synthesis. RcsA and RcsB form a DNA-binding transcriptional dual regulator. Deletion of this gene should in principle cause some repression of extracellular polysaccharides.[10]
BW25113 ΔfimH
FimH is the protein on the tip of fimbriae; they are the mannose sensitive subunit that in e coli mediate binding to receptor structures. [11]
BW25113 ΔcsgD
regulator of genes involved in curli assembly, induced in mid-exponential phase, csg-dependent genes active in stationary phase. [1]
BW25113 ΔompR
Function not clear. [12]
BW25113 ΔbcsA
Cellulose synthase, catalytic subunit. Its deletion should cause a lower cellulose content on the cell walls or an absence of cellulose, with a corresponding decrease in biofilm cell mass.[13]

WORKS CITED


[1] " Escherichia coli K-12 substr. MG1655 Polypeptide: CsgD DNA-binding transcriptional dual regulator," 2013. [Online]. Available: http://www.ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=G6546. [Accessed 27 Sept 2013].
[2] " Escherichia coli K-12 substr. MG1655 Polypeptide: regulator for fimA," [Online]. Available: http://www.ecocyc.org/ECOLI/NEW-IMAGE? type=GENE&object=EG10309. [Accessed 27 September 2013].
[3] " Escherichia coli K-12 substr. MG1655 Polypeptide: MlrA DNA binding transcriptional activator," [Online]. Available: http://www.ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG12008. [Accessed 27 September 2013].
[4] " Escherichia coli K-12 substr. MG1655 Polypeptide: outer membrane protein 3a (II*;G;d)," [Online]. Available: http://www.ecocyc.org/ECOLI/NEW-IMAGE? type=GENE&object=EG10669. [Accessed 27 September 2013].
[5] " Escherichia coli K-12 substr. MG1655 Enzyme: diguanylate cyclase," [Online]. Available: http://www.ecocyc.org/ECOLI/NEW-IMAGE? type=GENE&object=EG11643. [Accessed 27 September 2013].
[6] "Escherichia coli K-12 substr. MG1655 Polypeptide: major type 1 subunit fimbrin (pilin)," [Online]. Available: http://www.ecocyc.org/ECOLI/NEW-IMAGE? type=GENE&object=EG10308. [Accessed 27 September 2013].
[7] " Escherichia coli K-12 substr. MG1655 Enzyme: diguanylate cyclase," [Online]. Available: http://www.ecocyc.org/ECOLI/NEW-IMAGE? type=GENE&object=G6784. [Accessed 27 September 2013].
[8] "Escherichia coli K-12 substr. MG1655 Polypeptide: outer membrane protein X," [Online]. Available: http://www.ecocyc.org/ECOLI/NEW-IMAGE? type=GENE&object=EG12117. [Accessed 27 September 2013].
[9] "Escherichia coli K-12 substr. MG1655 Enzyme: poly-β-1,6-N-acetyl-D-glucosamine N-deacetylase," [Online]. Available: http://www.ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=G6530. [Accessed 27 September 2013].
[10] "Escherichia coli K-12 substr. MG1655 Polypeptide: positive DNA-binding transcriptional regulator of capsular polysaccharide synthesis, activates its own expression," [Online]. Available: http://www.ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG10820. [Accessed 27 September 2013].
[11] "Escherichia coli K-12 substr. MG1655 Polypeptide: minor fimbrial subunit, D-mannose specific adhesin," [Online]. Available: http://www.ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG10315. [Accessed 27 September 2013].
[12] "Escherichia coli K-12 substr. MG1655 Polypeptide: OmpR," [Online]. Available: http://www.ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG10672. [Accessed 27 September 2013].
[13] " Escherichia coli K-12 substr. MG1655 Polypeptide: cellulose synthase, catalytic subunit," [Online]. Available: http://www.ecocyc.org/ECOLI/NEW-IMAGE? type=GENE&object=EG12260. [Accessed 27 September 2013].

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 (insert reference here). 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. Inoculate 200 µL of 1:100 dilution of overnight culture along with 100 µL of 4%EtOH in buffered LB solution into a well in a 96-well plate to obtain a final EtOH concentration of 1%.
3. Inoculate 200 µL of 1:100 dilution of overnight culture along with 100 µL of 15%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 Ethanol 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. Inoculate 200 µL of 1:100 dilution of overnight culture along with 100 µL of
0.3%sucrose solutionmade in buffered LB solution into a well in a 96-well plate to obtain a final concentration of 0.1 M.
3. Inoculate 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 a negative biofilm effect. 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.