Team:Toronto/Project/Data

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<h7><font color=black><p style = "text-align:center; font-size:35px;"><b>DATA</b></p><br/>
<h7><font color=black><p style = "text-align:center; font-size:35px;"><b>DATA</b></p><br/>
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<p style = "font-size:18px;"><b>BW25113 WT</b><br/>
+
<p style = "font-size:18px;">
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<b>MG1655 WT</b>
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The following graphs show our relative spectrophotometer measurements for
-
<b>BW25113 pEBS-csgD</b><br/>
+
each assay axis when paired with each stimuli, for a specific strain. The 2
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<b>BW25113 pEBS-csgD</b><br/>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] <br/>
+
controls are wild type MG1655 and BW25113. The other strains tested have
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<b>BW25113 pEBS-fimB</b><br/>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]<br/>
+
been transformed with pEBS and induced with 0.3mM IPTG. <br/>
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<b>BW25113 pEBS-mlrA</b><br/>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]</br>
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<b>BW: BW25113</b><br/>
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<b>BW25113 pEBS-ompA </b><br/>The protein OmpA influences cellulose production by
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<b>MG: MG1655</b><br/>
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repressing cellulose production with CpxRA stress response system; it is
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<b>BWC: BW25113 WITH csgD</b><br/>
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overexpressed in biofilm formation and repressed when cells are exposed to
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<b>BWF: BW25113 WITH fimB</b><br/>
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visible light. [4]</br>
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<b>BWM: BW25113 WITH mlrA</b><br/>
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<b>BW25113 pEBS-ydeH </b><br/>YdeH is a diguanylate cyclase, where its product
+
<b>BWO: BW25113 WITH ompA</b><br/>
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regulates biofilm formation and motility. Its deletion has been shown to reduce
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<b>BWY: BW25113 WITH csgD</b><br/>
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surface attachment of the cell, its overexpression has been shown to reduce
+
-
motility as well as flagella. [5]<br/>
+
-
<b>BW25113 ΔfimA </b><br/>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]<br/>
+
-
<b>BW25113 ΔompA</b><br/>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]<br/>
+
-
<b>BW25113 ΔdosC </b><br/>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]<br/>
+
-
<b>BW25113 ΔompX </b><br/>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]<br/>
+
-
<b> BW25113 ΔpgaB</b><br/>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] <br/>
+
-
<b>BW25113 ΔrcsA </b><br/>+ 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]<br/>
+
-
<b>BW25113 ΔfimH</b><br/>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] <br/>
+
-
<b>BW25113 ΔcsgD</b><br/>regulator of genes involved in curli assembly, induced in
+
-
mid-exponential phase, csg-dependent genes active in stationary phase. [1] <br/>
+
-
<b>BW25113 ΔompR</b><br/>Function not clear. [12]<br/>
+
-
<b>BW25113 ΔbcsA </b><br/>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]</br>
+
-
<p style = "font-size:35px;"><b>WORKS CITED</b></p><br/>
+
-
[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].<br/>
 
-
[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].<br/>
 
-
[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].<br/>
 
-
[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].<br/>
 
-
[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].<br/>
 
-
[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].<br/>
 
-
[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].<br/>
 
-
[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].<br/>
 
-
[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].<br/>
 
-
[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].<br/>
 
-
[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].<br/>
 
-
[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].<br/>
 
-
[13] " Escherichia coli K-12 substr. MG1655 Polypeptide: cellulose synthase, catalytic
 
-
subunit," [Online]. Available: http://www.ecocyc.org/ECOLI/NEW-IMAGE?
 
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type=GENE&object=EG12260. [Accessed 27 September 2013].<br/>
 
-
<font color=black><p style = "text-align:center; font-size:35px;"><b>STIMULI PROTOCOLS</b></p><br/>
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See .pdf <a href="https://static.igem.org/mediawiki/2013/2/2d/Produced_by_convert-jpg-to-pdf.net.pdf">here</a>
-
<p style = "font-size:18px;"><b><u>Prelude</u></b><br/>
+
-
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.<br>
+
-
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.<br/>
+
-
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.<br/>
+
-
In continuation with the project, we are planning to optimize results by combining the treatments
+
-
which give the best results. <br/>
+
-
<p style = "font-size:18px;"><b><u>Ethanol</u></b><br/>
+
-
Reasoning:</br>
+
-
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%.</br>
+
-
Materials for Ethanol stimulus:</br>
+
-
• Overnight cultures of E. coli<br/>
+
-
•40% EtOH solution prepared in phosphate buffered LB<br/>
+
-
• 96-well plate<br/>
+
-
<p style = "font-size:18px;"><b>Protocol:</b><br/>
+
-
1. Grow overnight culture of E. coli in phosphate buffered media.<br/>
+
-
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%.<br/>
+
-
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%.<br/>
+
-
4. Incubate in darkness at 23 degrees C for 48 hours.<br/>
+
-
Lim <i>et al.</i> (2012) Dimethyl sulfoxide and ethanol elicit increased amyloid biogenesis and
+
-
amyloid-integrated biofilm formation in Escherichia coli. <i>Appl Environ Microbiol</i> <b>78</b>:3369-78.
+
-
<p style = "text-align:center; font-size:18px;"><b>Carbon Source</b><br/>
+
-
<p style = "font-size:18px;"><b><u>Sucrose</u></b><br/>
+
-
Reasoning:<br/>
+
-
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.<br/>
+
-
Materials for Ethanol stimulus:<br/>
+
-
• Overnight cultures of E. coli<br/>
+
-
• 2 M sucrose stock solution prepared in phosphate buffered LB<br/>
+
-
• 96-well plate<br/>
+
-
Protocol:<br/>
+
-
1. Grow overnight cultures in phosphate buffered media.<br/>
+
-
2. Inoculate 200 µL of 1:100 dilution of overnight culture along with 100 µL of <br/>
+
-
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.<br/>
+
-
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.<br/>
+
-
4. Incubate in darkness at 23 degrees C for 48 hours.<br/>
+
-
Kawarai <i>et al. </i>(2009) Biofilm formation by Escherichia coli in hypertonic sucrose media. <i>J
+
-
Biosci Bioeng</i> <b>107:</b>630-5.<br/>
+
-
<p style = "font-size:18px;"><b><u>Indole</u></b><br/>
 
-
Reasoning:<br/>
 
-
Indole has a negative biofilm effect. The concentrations being tested are 500 micromolar and 300
 
-
micromolar.<br/>
 
-
Materials:<br/>
 
-
• 0.0009 M Indole in phosphate buffered LB<br/>
 
-
• Overnight culture diluted in a 1:100 ratio<br/>
 
-
Protocol:<br/>
 
-
1. 300 µM<br/>
 
-
&nbsp;&nbsp;a. 1:100 dilution of overnight culture was inoculated in buffered LB.<br/>
 
-
&nbsp;&nbsp;b. 200 µl of the dilution was added to the well.<br/>
 
-
&nbsp;&nbsp;c. 100 µl of 0.0009 M indole was added to the well.<br/>
 
-
&nbsp;&nbsp;d. The culture was incubated for 48 hours at 23 Celsius. <br/>
 
-
2. 500 µM<br/>
 
-
&nbsp;&nbsp;a. 1:100 dilution of overnight culture was incubated in buffered LB.<br/>
 
-
&nbsp;&nbsp;b. 200 µl of the dilution was added to the well.<br/>
 
-
&nbsp;&nbsp;c. 100 µl of 0.0015 M was added to the well.<br/>
 
-
&nbsp;&nbsp;d. The culture was incubated for 48 hours at 23 Celsius.<br/>
 
-
Bansal <i>et al.</i> (2007) Differential effects of epinephrine, norepinephrine, and indole on
 
-
Escherichia coli O157:H7 chemotaxis, colonization, and gene expression.<i> Infect
 
-
Immun</i> <b>75</b>:4597-607.
 
-
 
-
<p style = "font-size:18px;"><b><u>DMSO</u></b><br/>
 
-
Reasoning:</br>
 
-
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%.<br/>
 
-
Materials list:<br/>
 
-
• Dimethyl sulfoxide in phosphate buffered LB<br/>
 
-
• Overnight culture diluted in a 1:100 ratio<br/>
 
-
Protocol:<br/>
 
-
1. 2%<br/>
 
-
&nbsp;&nbsp;a. 1:100 dilution of overnight culture was incubated in buffered LB.<br/>
 
-
&nbsp;&nbsp;b. 200 µl of the dilution was added to the well.<br/>
 
-
&nbsp;&nbsp;c. 100 µl of 6% DMSO was added to the well.<br/>
 
-
&nbsp;&nbsp;d. The culture was incubated for 48 hours at 23 Celsius. <br/>
 
-
2. 4%<br/>
 
-
&nbsp;&nbsp;a. 1:100 dilution of overnight culture was incubated in buffered LB.<br/>
 
-
&nbsp;&nbsp;b. 200 µl of the dilution was added to the well.<br/>
 
-
&nbsp;&nbsp;c. 100 µl of 12% DMSO was added to the well.<br/>
 
-
&nbsp;&nbsp;d. The culture was incubated for 48 hours at 23 degrees Celsius<br/>
 
-
Lim <i>et al.</i> (2012) Dimethyl sulfoxide and ethanol elicit increased amyloid biogenesis and
 
-
amyloid-integrated biofilm formation in Escherichia coli. <i>Appl Environ Microbiol</i> <b>78</b>:3369-78.<br/>
 
-
<p style = "text-align:center; font-size:18px;"><b>Media</b><br/>
 
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<p style = "font-size:18px;"><b><u>NaCl stimulus</u></b><br/>
 
-
Reasoning:<br/>
 
-
The concentrations being tested are 0.3 M and 0.5 M. NaCl is being tested as an ionic solute.
 
-
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.<br/>
 
-
Materials:<br/>
 
-
• Phosphate buffered LB without NaCl<br/>
 
-
• Overnight culture diluted in a 1:100 ratio<br/>
 
-
Protocol:<br/>
 
-
1. Sodium chloride free Luria Broth<br/>
 
-
&nbsp;&nbsp;a. 275 µl of NaCl-free LB was added to the well.<br/>
 
-
&nbsp;&nbsp;b. 25 µl of overnight culture was added to the well.<br/>
 
-
&nbsp;&nbsp;c. The culture was incubated for 48 hours at 23 degrees Celsius.<br/>
 
-
Yeom <i>et al.</i> (2012) Effects of non-ionic solute stresses on biofilm formation and
 
-
lipopolysaccharide production in Escherichia coli O157:H7. <i>Res Microbiol</i> <b>163</b>:258-67<br/>
 
-
<p style = "font-size:18px;"><b><u>Terrific Broth (TB)</u></b><br/>
 
-
Reasoning:<br/>
 
-
Verma et al. (2010) found the least amount of biofilm to be produced at 37 degrees in TB.</br>
 
-
Materials:<br/>
 
-
• 1:100 dilution of overnight culture grown in phosphate buffered media</br>
 
-
Protocol:<br/>
 
-
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.<br/>
 
-
2. Add 100 mL of a sterile solution containing a final concentration of 0.17 M KH2PO4, 0.72
 
-
M K2HPO4.<br/>
 
-
3. Autoclave again.. Add 275 µl of Terrific Broth to the well.<br/>
 
-
5. Add 25 µl of overnight culture to the well.<br/>
 
-
6. Incubate the culture for 48 hours at 23 degrees.<br/>
 
-
Prüss <i>et al.</i> (2010) Environmental and genetic factors that contribute to Escherichia coli K-12
 
-
biofilm formation. <i>Arch Microbiol</i> <b>192</b>:715-28.<br/>
 
-
 
-
<p style = "font-size:18px;"><b><u>Tryptone Soy Broth</u></b><br/>
 
-
Reasoning:<br/>
 
-
Verma et al. (2010) found most biofilm to be produced at 37 degrees in TSB media.<br/>
 
-
Materials:<br/>
 
-
• TSB media<br/>
 
-
• Overnight culture<br/>
 
-
Protocol:<br/>
 
-
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.<br/>
 
-
2. 275 µl of Tryptic Soy Broth was added to the well.<br/>
 
-
3. 25 µl of overnight culture was added to the well.<br/>
 
-
4. The culture was incubated for 48 hours at 23 degrees.<br/>
 
</p>
</p>
</font>
</font>

Latest revision as of 03:09, 28 September 2013

DATA


The following graphs show our relative spectrophotometer measurements for each assay axis when paired with each stimuli, for a specific strain. The 2 controls are wild type MG1655 and BW25113. The other strains tested have been transformed with pEBS and induced with 0.3mM IPTG.
BW: BW25113
MG: MG1655
BWC: BW25113 WITH csgD
BWF: BW25113 WITH fimB
BWM: BW25113 WITH mlrA
BWO: BW25113 WITH ompA
BWY: BW25113 WITH csgD
See .pdf here




















































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