Difference between revisions of "Team:UofGuelph/Description"

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<body">
  
−
<h1 class="descHead">Project Description</h1>
+
<h1 class="descHead">Project Design</h1>
 
<img src="https://static.igem.org/mediawiki/2017/4/4a/T--
 
<img src="https://static.igem.org/mediawiki/2017/4/4a/T--
  
 
U_of_Guelph--gryphon.jpg" class="guelphImages">
 
U_of_Guelph--gryphon.jpg" class="guelphImages">
 +
<h1 class="descSub">Whats the Deal with Beerstone?</h1>
 +
<p class="descP">
 +
Beerstone can form on any surface that comes into contact
  
−
<h1 class="descSub">Background on Beerstone, FRC and  
+
with beer and wort (unfermented beer) and has been a
  
−
OXC</h1>
+
problem for brewers as long as beer has been
−
<p class="descP">
+
−
Beerstone is a salt precipitate composed primarily of
+
  
−
calcium oxalate (C<sub>2</sub>CaO<sub>4</sub>). It poses
+
produced<sup>1, 2</sup>. The most problematic locations
  
−
a large problem in the brewing industry due to its high
+
for its formation are heat exchangers, fermentation
  
−
insolubility and use of corrosive chemicals for its
+
vessels, aging tanks, kegs, and beer dispense lines. 
  
−
effective removal from brewing equipment<sup>1</sup>.
+
Beerstone is comprised of a combination of precipitated
−
<br><br>
+
−
The reason for the high insolubility of beerstone is  
+
  
−
because one of its major components, calcium oxalate  
+
calcium oxalate and entrapped beer
  
−
(C<sub>2</sub>CaO<sub>4</sub>), contains a chelator.  
+
polypeptides<sup>3</sup>. Oxalate enters the brewing
  
−
Calcium ions in the water react with oxalic acids present
+
process from the cereal grains and hops used to make
  
−
in malt, forming calcium oxalate. When polypeptides found
+
beer<sup>4</sup>. Oxalate is present in the form of
  
−
in beer are incorporated into the oxalate structure, the
+
aqueous oxalic acid which is a corrosive, highly oxidized
  
−
precipitate that is formed is known as
+
compound that has strong chelating activity<sup>5</sup>.
  
−
beerstone<sup>2,3</sup>. Geographic regions that contain
+
These oxalate ions are soluble in both wort and beer,  
  
−
high levels of calcium in their drinking water, such as
+
allowing them to combine with calcium ions to form
  
−
Guelph, Ontario, Canada, can lead to 165g of
+
calcium oxalate<sup>4</sup>. Calcium ions enter the
  
−
C<sub>2</sub>CaO<sub>4</sub> buildup per 1000L batch of
+
brewing process through the water, grains and water-
  
−
beer<sup>4</sup>.  
+
correction salts<sup>4</sup>. Calcium oxalate
−
The porous nature of beerstone scale promotes biofilm
+
  
−
formation from the microorganisms present in the brewing
+
precipitates out of solution upon formation, and is one
  
−
solution. Biofilm growth causes both “off flavours” that
+
of the most insoluble metallo-organic compounds with a
  
−
can ruin an entire batch of beer and also be a potential
+
low solubility<sup>4</sup>. In a geographic region with
  
−
biosafety hazard for the consumer<sup>5</sup>.
+
high calcium levels in the drinking water, such as
−
<br><br>
+
−
<i>Oxalobacter formigenes</i> is a human gut bacterium
+
  
−
that derives its energy solely from the metabolization of  
+
Guelph, Ontario, this can lead to up to 165g of calcium
  
−
oxalate using enzymes Formyl-Coenzyme A Transferase (FRC)
+
oxalate building up in a single 1000L batch of
  
−
and Oxalyl-Coenzyme A Decarboxylase (OXC). Oxalate is
+
beer<sup>1</sup>. This precipitation can happen either
  
−
brought into the cell by an oxalate-formate antiporter
+
during the brewing or bottling processes, or after the  
  
−
(OxIT) and converted to CO2 and formyl-CoA. The formyl-
+
beer has been bottled depending on when the calcium
  
−
CoA is reused by FRC as a CoA donor in a subsequent
+
oxalate formation occurs<sup>4</sup>. The point during
  
−
reaction and released from the cell as formate by
+
the process at which calcium oxalate forms is dependent
  
−
OxIT<sup>6</sup>.
+
on temperature, time, pH and ion
−
</p>
+
  
−
<h1 class="descSub">Objectives</h1>
+
concentration<sup>4</sup>. If calcium oxalate forms after
−
<p class="descP">
+
 
−
- Express FRC and OXC in <i>E. coli</i> BL21 using pET28a
+
filtration, or if all calcium oxalate crystals are not
 +
 
 +
filtered out of the beer, haze and sediment may
 +
 
 +
form<sup>4</sup>. In addition, calcium oxalate crystals
 +
 
 +
can cause over-foaming during filling or when a
 +
 
 +
pressurized container such as a can or bottle is
 +
 
 +
opened<sup>4</sup>.<br><br>
 +
Deposits of beerstone provide protection and nutrients
 +
 
 +
for bacteria to grow due to the porous surface of the
 +
 
 +
beerstone, into which nutrient-providing proteins often
 +
 
 +
become entrapped<span class="super">6</span>. This allows
 +
 
 +
for unwanted microbial growth and the formation of
 +
 
 +
biofilms upon the beerstone, for example, species of the
 +
 
 +
genera <i>Pectinatus</i> and <i>Megasphaera</i>. These
 +
 
 +
microorganisms cause beer spoilage, products with reduced
 +
 
 +
shelf-life, off-flavours and sour tastes, rendering the
 +
 
 +
beer unsuitable for sale or consumption thus resulting in
 +
 
 +
financial loss to the brewer<span class="super">7</span>.
 +
 
 +
<i>Lactobacillus</i> species in beer, in particular,
 +
 
 +
causes high turbidity which manifests as a hazy
 +
 
 +
appearance in the liquid. It also causes a high level of
 +
 
 +
diacetyl in the beer, resulting in an unwanted ‘buttery’
 +
 
 +
flavour<span class="super">7</span>. With the removal of
 +
 
 +
beerstone, growth of these microbial contaminants will be
 +
 
 +
prohibited, improving brew quality and reducing
 +
 
 +
downstream processing. <br><br>
 +
Beerstone is difficult to remove for several reasons.
 +
 
 +
Calcium oxalate is extremely insoluble in both hot and
 +
 
 +
cold water, meaning that the use of harsh chemicals is
 +
 
 +
currently required for effective methods of removing
 +
 
 +
beerstone. These involve caustic or other harsh cleaners
 +
 
 +
which are dangerous to work with due to the potential for
 +
 
 +
exposure burns of the eyes and skin, and corrosive damage
 +
 
 +
to surfaces<span class="super">2, 3, 8</span>. The use of
 +
 
 +
these cleaning agents require long and frequent pauses in
 +
 
 +
production which lower the efficacy of the brewing
 +
 
 +
process. Additionally, the equipment required to utilize
 +
 
 +
these caustic chemicals and the chemical disposal
  
−
vector.
+
requirements are costly and potentially environmentally
−
- Assess the feasibility of using these enzymes as an
+
  
−
alternate cleaning method to degrade beerstone.
+
damaging.  
 
</p>
 
</p>
  
−
<h1 class="descSub">Project Overview</h1>
+
<h1 class="descSub"><i>Oxalobacter formigenes</i> and the
  
 +
Breakdown of Oxalate</h1>
 
<p class="descP">
 
<p class="descP">
−
<b>Step 1: Cloning of <i>frc</i> and <i>oxc</i> into DH5α
+
<i>Oxalobacter formigenes</i> is an anaerobic, Gram-
  
−
</b><br>
+
negative bacteria native to the human gut microbiota<span
−
-Synthesize <i>frc</i> and <i>oxc</i><br>
+
−
-Add PstI cut site to pET-28a <br>
+
−
-Ligate <i>frc</i> and <i>oxc</i> into pET-28a <br>
+
−
-Transform pET-28afrc/oxc into DH5α <br>
+
−
<br>
+
−
<b>Step 2: Clone <i>frc</i> and <i>oxc</i> into
+
  
−
BL21</b><br>
+
class="super">5</span>.  <i>O. formigenes</i> is a safe
−
-Purify pET-28afrc/oxc from DH5α<br>
+
−
-Transform pET-28afrc/oxc into BL21<br>
+
−
<br>
+
−
<b>Step 3: Express and Purify FRC and OXC</b><br>
+
−
-Induce expression with IPTG and extract crude
+
  
−
proteins<br>
+
(biosafety hazard level 1) organism that relies solely on  
−
-Purify proteins using Ni-NTA chromatography<br>
+
−
<br>
+
−
<b>Step 4: Characterize FRC and OXC</b><br>
+
−
-Characterize enzyme function using Sodium Oxalate<br>
+
−
-Characterize enzyme function using Calcium Oxalate<br>
+
−
<br>
+
−
<b>Step 5: Design a Cleaning Solution and Test on  
+
  
−
Beerstone</b><br>
+
oxalate as its source of energy, as well as its main
−
-Test ability of enzymes to break down Beerstone<br>
+
−
-Design a functional cleaning solution<br>
+
  
 +
source of carbon<span class="super">5, 9</span>. 
 +
 +
Metabolism of oxalate is accomplished by two enzymes,
 +
 +
Formyl Coenzyme A Transferase (FRC) and Oxalyl-Coenzyme A
 +
 +
Decarboxylase (OXC)<span class="super">5, 10</span>.
 +
In the first reaction step, a Coenzyme A (CoA) is
 +
 +
transferred to oxalate by the FRC enzyme<span
 +
 +
class="super">10</span>.  It functions by forming a
 +
 +
ternary complex between the substrates and the enzyme,
 +
 +
resulting in an oxalyl-CoA complex<span class="super">5,
 +
 +
10</span>.  The OXC enzyme then catalyzes a reductive
 +
 +
reaction in which formyl-CoA and CO2 are produced<span
 +
 +
class="super">5, 10</span>.  The FRC enzyme then cycles
 +
 +
the CoA back to a new oxalate molecule to start the
 +
 +
process again<span class="super">5, 10</span>.  Thiamine
 +
 +
pyrophosphate (TPP), Mg2+, acetate, and CoA are required
 +
 +
for the reaction to take place<span class="super">5,
 +
 +
10</span>.  The reaction mechanism for the breakdown of
 +
 +
calcium oxalate by FRC and OXC can be shown by the
 +
 +
equations in Figure 1<span class="super">10</span>:
 +
<br><br>
 
</p>
 
</p>
  
 +
<h1 class="descSub">Our Project Design</h1>
 +
 +
<p class="descP"> Here we will include information about
 +
 +
why we plan on doing our project the way we are, what our
 +
 +
plans are in some detail, and why we are doing what we
 +
 +
are doing this year<br><br>To be, or not to be: that is
 +
 +
the question:
 +
Whether ’tis nobler in the mind to suffer
 +
The slings and arrows of outrageous fortune,
 +
Or to take arms against a sea of troubles,
 +
And by opposing end them? To die: to sleep;
 +
No more; and by a sleep to say we end
 +
The heart-ache and the thousand natural shocks
 +
That flesh is heir to, ’tis a consummation
 +
Devoutly to be wish’d. To die, to sleep;
 +
To sleep: perchance to dream: ay, there’s the rub;
 +
For in that sleep of death what dreams may come
 +
When we have shuffled off this mortal coil,
 +
Must give us pause: there’s the respect
 +
That makes calamity of so long life;
 +
For who would bear the whips and scorns of time,
 +
The oppressor’s wrong, the proud man’s contumely,
 +
The pangs of despised love, the law’s delay,
 +
The insolence of office and the spurns
 +
That patient merit of the unworthy takes,
 +
When he himself might his quietus make
 +
With a bare bodkin? who would fardels bear,
 +
To grunt and sweat under a weary life,
 +
But that the dread of something after death,
 +
The undiscover’d country from whose bourn
 +
No traveller returns, puzzles the will
 +
And makes us rather bear those ills we have
 +
Than fly to others that we know not of?
 +
Thus conscience does make cowards of us all;
 +
And thus the native hue of resolution
 +
Is sicklied o’er with the pale cast of thought,
 +
And enterprises of great pith and moment
 +
With this regard their currents turn awry,
 +
And lose the name of action.–Soft you now!
 +
The fair Ophelia! Nymph, in thy orisons
 +
Be all my sins remember’d.</p>
 +
 +
<h1 class="descSub">Future Development</h1>
 +
<p class="descP"> Here we will write about our plans for
 +
 +
the future of this project and what direction we plan on
 +
 +
heading. <br><br>To be, or not to be: that is the
 +
 +
question:
 +
Whether ’tis nobler in the mind to suffer
 +
The slings and arrows of outrageous fortune,
 +
Or to take arms against a sea of troubles,
 +
And by opposing end them? To die: to sleep;
 +
No more; and by a sleep to say we end
 +
The heart-ache and the thousand natural shocks
 +
That flesh is heir to, ’tis a consummation
 +
Devoutly to be wish’d. To die, to sleep;
 +
To sleep: perchance to dream: ay, there’s the rub;
 +
For in that sleep of death what dreams may come
 +
When we have shuffled off this mortal coil,
 +
Must give us pause: there’s the respect
 +
That makes calamity of so long life;
 +
For who would bear the whips and scorns of time,
 +
 +
<h1 class="descSub">References</h1>
 +
<p class="descRef"> Rose, D. This is a test (2017). Sci.
 +
 +
Awesome. 28-29 </p>
  
 
<div class="sponsor">
 
<div class="sponsor">
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</body>
 
</body>
 +
 +
 +
<div class="column full_size">
 +
<h1>Design</h1>
 +
<p>
 +
Design is the first step in the design-build-test cycle
 +
 +
in engineering and synthetic biology. Use this page to
 +
 +
describe the process that you used in the design of your
 +
 +
parts. You should clearly explain the engineering
 +
 +
principles used to design your project.
 +
</p>
 +
 +
<p>
 +
This page is different to the "Applied Design Award"
 +
 +
page. Please see the <a
 +
 +
href="https://2018.igem.org/Team:UofGuelph/Applied_Design"
 +
 +
>Applied Design</a> page for more information on how to
 +
 +
compete for that award.
 +
</p>
 +
 +
</div>
 +
 +
 +
 +
<div class="column two_thirds_size">
 +
<h3>What should this page contain?</h3>
 +
<ul>
 +
<li>Explanation of the engineering principles your team
 +
 +
used in your design</li>
 +
<li>Discussion of the design iterations your team went
 +
 +
through</li>
 +
<li>Experimental plan to test your designs</li>
 +
</ul>
 +
 +
</div>
 +
 +
 +
 +
 
</html>
 
</html>

Revision as of 23:28, 6 December 2018

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Project Design

Whats the Deal with Beerstone?

Beerstone can form on any surface that comes into contact with beer and wort (unfermented beer) and has been a problem for brewers as long as beer has been produced1, 2. The most problematic locations for its formation are heat exchangers, fermentation vessels, aging tanks, kegs, and beer dispense lines. Beerstone is comprised of a combination of precipitated calcium oxalate and entrapped beer polypeptides3. Oxalate enters the brewing process from the cereal grains and hops used to make beer4. Oxalate is present in the form of aqueous oxalic acid which is a corrosive, highly oxidized compound that has strong chelating activity5. These oxalate ions are soluble in both wort and beer, allowing them to combine with calcium ions to form calcium oxalate4. Calcium ions enter the brewing process through the water, grains and water- correction salts4. Calcium oxalate precipitates out of solution upon formation, and is one of the most insoluble metallo-organic compounds with a low solubility4. In a geographic region with high calcium levels in the drinking water, such as Guelph, Ontario, this can lead to up to 165g of calcium oxalate building up in a single 1000L batch of beer1. This precipitation can happen either during the brewing or bottling processes, or after the beer has been bottled depending on when the calcium oxalate formation occurs4. The point during the process at which calcium oxalate forms is dependent on temperature, time, pH and ion concentration4. If calcium oxalate forms after filtration, or if all calcium oxalate crystals are not filtered out of the beer, haze and sediment may form4. In addition, calcium oxalate crystals can cause over-foaming during filling or when a pressurized container such as a can or bottle is opened4.

Deposits of beerstone provide protection and nutrients for bacteria to grow due to the porous surface of the beerstone, into which nutrient-providing proteins often become entrapped6. This allows for unwanted microbial growth and the formation of biofilms upon the beerstone, for example, species of the genera Pectinatus and Megasphaera. These microorganisms cause beer spoilage, products with reduced shelf-life, off-flavours and sour tastes, rendering the beer unsuitable for sale or consumption thus resulting in financial loss to the brewer7. Lactobacillus species in beer, in particular, causes high turbidity which manifests as a hazy appearance in the liquid. It also causes a high level of diacetyl in the beer, resulting in an unwanted ‘buttery’ flavour7. With the removal of beerstone, growth of these microbial contaminants will be prohibited, improving brew quality and reducing downstream processing.

Beerstone is difficult to remove for several reasons. Calcium oxalate is extremely insoluble in both hot and cold water, meaning that the use of harsh chemicals is currently required for effective methods of removing beerstone. These involve caustic or other harsh cleaners which are dangerous to work with due to the potential for exposure burns of the eyes and skin, and corrosive damage to surfaces2, 3, 8. The use of these cleaning agents require long and frequent pauses in production which lower the efficacy of the brewing process. Additionally, the equipment required to utilize these caustic chemicals and the chemical disposal requirements are costly and potentially environmentally damaging.

Oxalobacter formigenes and the Breakdown of Oxalate

Oxalobacter formigenes is an anaerobic, Gram- negative bacteria native to the human gut microbiota5. O. formigenes is a safe (biosafety hazard level 1) organism that relies solely on oxalate as its source of energy, as well as its main source of carbon5, 9. Metabolism of oxalate is accomplished by two enzymes, Formyl Coenzyme A Transferase (FRC) and Oxalyl-Coenzyme A Decarboxylase (OXC)5, 10. In the first reaction step, a Coenzyme A (CoA) is transferred to oxalate by the FRC enzyme10. It functions by forming a ternary complex between the substrates and the enzyme, resulting in an oxalyl-CoA complex5, 10. The OXC enzyme then catalyzes a reductive reaction in which formyl-CoA and CO2 are produced5, 10. The FRC enzyme then cycles the CoA back to a new oxalate molecule to start the process again5, 10. Thiamine pyrophosphate (TPP), Mg2+, acetate, and CoA are required for the reaction to take place5, 10. The reaction mechanism for the breakdown of calcium oxalate by FRC and OXC can be shown by the equations in Figure 110:

Our Project Design

Here we will include information about why we plan on doing our project the way we are, what our plans are in some detail, and why we are doing what we are doing this year

To be, or not to be: that is the question: Whether ’tis nobler in the mind to suffer The slings and arrows of outrageous fortune, Or to take arms against a sea of troubles, And by opposing end them? To die: to sleep; No more; and by a sleep to say we end The heart-ache and the thousand natural shocks That flesh is heir to, ’tis a consummation Devoutly to be wish’d. To die, to sleep; To sleep: perchance to dream: ay, there’s the rub; For in that sleep of death what dreams may come When we have shuffled off this mortal coil, Must give us pause: there’s the respect That makes calamity of so long life; For who would bear the whips and scorns of time, The oppressor’s wrong, the proud man’s contumely, The pangs of despised love, the law’s delay, The insolence of office and the spurns That patient merit of the unworthy takes, When he himself might his quietus make With a bare bodkin? who would fardels bear, To grunt and sweat under a weary life, But that the dread of something after death, The undiscover’d country from whose bourn No traveller returns, puzzles the will And makes us rather bear those ills we have Than fly to others that we know not of? Thus conscience does make cowards of us all; And thus the native hue of resolution Is sicklied o’er with the pale cast of thought, And enterprises of great pith and moment With this regard their currents turn awry, And lose the name of action.–Soft you now! The fair Ophelia! Nymph, in thy orisons Be all my sins remember’d.

Future Development

Here we will write about our plans for the future of this project and what direction we plan on heading.

To be, or not to be: that is the question: Whether ’tis nobler in the mind to suffer The slings and arrows of outrageous fortune, Or to take arms against a sea of troubles, And by opposing end them? To die: to sleep; No more; and by a sleep to say we end The heart-ache and the thousand natural shocks That flesh is heir to, ’tis a consummation Devoutly to be wish’d. To die, to sleep; To sleep: perchance to dream: ay, there’s the rub; For in that sleep of death what dreams may come When we have shuffled off this mortal coil, Must give us pause: there’s the respect That makes calamity of so long life; For who would bear the whips and scorns of time,

References

Rose, D. This is a test (2017). Sci. Awesome. 28-29

University of Guelph iGEM 2018

Design

Design is the first step in the design-build-test cycle in engineering and synthetic biology. Use this page to describe the process that you used in the design of your parts. You should clearly explain the engineering principles used to design your project.

This page is different to the "Applied Design Award" page. Please see the Applied Design page for more information on how to compete for that award.

What should this page contain?

  • Explanation of the engineering principles your team used in your design
  • Discussion of the design iterations your team went through
  • Experimental plan to test your designs