Revision as of 23:28, 6 December 2018

Project Description

Background on Beerstone, FRC and OXC

Beerstone is a salt precipitate composed primarily of calcium oxalate (C₂CaO₄). It poses a large problem in the brewing industry due to its high insolubility and use of corrosive chemicals for its effective removal from brewing equipment¹.

The reason for the high insolubility of beerstone is because one of its major components, calcium oxalate (C₂CaO₄), contains a chelator. Calcium ions in the water react with oxalic acids present in malt, forming calcium oxalate. When polypeptides found in beer are incorporated into the oxalate structure, the precipitate that is formed is known as beerstone^2,3. Geographic regions that contain high levels of calcium in their drinking water, such as Guelph, Ontario, Canada, can lead to 165g of C₂CaO₄ buildup per 1000L batch of beer⁴. The porous nature of beerstone scale promotes biofilm formation from the microorganisms present in the brewing solution. Biofilm growth causes both “off flavours” that can ruin an entire batch of beer and also be a potential biosafety hazard for the consumer⁵.

Oxalobacter formigenes is a human gut bacterium that derives its energy solely from the metabolization of oxalate using enzymes Formyl-Coenzyme A Transferase (FRC) and Oxalyl-Coenzyme A Decarboxylase (OXC). Oxalate is brought into the cell by an oxalate-formate antiporter (OxIT) and converted to CO2 and formyl-CoA. The formyl-CoA is reused by FRC as a CoA donor in a subsequent reaction and released from the cell as formate by OxIT⁶.

Objectives

- Express FRC and OXC in E. coli BL21 using pET28a vector. - Assess the feasibility of using these enzymes as an alternate cleaning method to degrade beerstone.

Project Overview

Step 1: Cloning of frc and oxc into DH5α
-Synthesize frc and oxc
-Add PstI cut site to pET-28a
-Ligate frc and oxc into pET-28a
-Transform pET-28afrc/oxc into DH5α

Step 2: Clone frc and oxc into BL21
-Purify pET-28afrc/oxc from DH5α
-Transform pET-28afrc/oxc into BL21

Step 3: Express and Purify FRC and OXC
-Induce expression with IPTG and extract crude proteins
-Purify proteins using Ni-NTA chromatography

Step 4: Characterize FRC and OXC
-Characterize enzyme function using Sodium Oxalate
-Characterize enzyme function using Calcium Oxalate

Step 5: Design a Cleaning Solution and Test on Beerstone
-Test ability of enzymes to break down Beerstone
-Design a functional cleaning solution

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- }
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- vertical-align: super;
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- }
 </style>
-<body">
-<h1 class="descHead">Project Design</h1>
+<h1 class="descHead">Project Description</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">Background on Beerstone, FRC and OXC</h1>
-<h1 class="descSub">Whats the Deal with Beerstone?</h1>
 <p class="descP">
-Beerstone can form on any surface that comes into contact
+Beerstone is a salt precipitate composed primarily of calcium oxalate (C<sub>2</sub>CaO<sub>4</sub>). It poses a large problem in the brewing industry due to its high insolubility and use of corrosive chemicals for its effective removal from brewing equipment<sup>1</sup>.
+<br><br>
+The reason for the high insolubility of beerstone is because one of its major components, calcium oxalate (C<sub>2</sub>CaO<sub>4</sub>), contains a chelator. Calcium ions in the water react with oxalic acids present in malt, forming calcium oxalate. When polypeptides found in beer are incorporated into the oxalate structure, the precipitate that is formed is known as beerstone<sup>2,3</sup>. Geographic regions that contain high levels of calcium in their drinking water, such as Guelph, Ontario, Canada, can lead to 165g of C<sub>2</sub>CaO<sub>4</sub> buildup per 1000L batch of beer<sup>4</sup>.
+The porous nature of beerstone scale promotes biofilm formation from the microorganisms present in the brewing solution. Biofilm growth causes both “off flavours” that can ruin an entire batch of beer and also be a potential biosafety hazard for the consumer<sup>5</sup>.
+<br><br>
+<i>Oxalobacter formigenes</i> is a human gut bacterium that derives its energy solely from the metabolization of oxalate using enzymes Formyl-Coenzyme A Transferase (FRC) and Oxalyl-Coenzyme A Decarboxylase (OXC). Oxalate is brought into the cell by an oxalate-formate antiporter (OxIT) and converted to CO2 and formyl-CoA. The formyl-CoA is reused by FRC as a CoA donor in a subsequent reaction and released from the cell as formate by OxIT<sup>6</sup>.
+ </p>
-with beer and wort (unfermented beer) and has been a
+<h1 class="descSub">Objectives</h1>
+<p class="descP">
-problem for brewers as long as beer has been
+- Express FRC and OXC in <i>E. coli</i> BL21 using pET28a vector.
+- Assess the feasibility of using these enzymes as an alternate cleaning method to degrade beerstone.
-produced<sup>1, 2</sup>. 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
-polypeptides<sup>3</sup>. Oxalate enters the brewing
-process from the cereal grains and hops used to make
-beer<sup>4</sup>. Oxalate is present in the form of
-aqueous oxalic acid which is a corrosive, highly oxidized
-compound that has strong chelating activity<sup>5</sup>.
-These oxalate ions are soluble in both wort and beer,
-allowing them to combine with calcium ions to form
-calcium oxalate<sup>4</sup>. Calcium ions enter the
-brewing process through the water, grains and water-
-correction salts<sup>4</sup>. Calcium oxalate
-precipitates out of solution upon formation, and is one
-of the most insoluble metallo-organic compounds with a
-low solubility<sup>4</sup>. 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
-beer<sup>1</sup>. 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 occurs<sup>4</sup>. The point during
-the process at which calcium oxalate forms is dependent
-on temperature, time, pH and ion
-concentration<sup>4</sup>. If calcium oxalate forms after
-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
-requirements are costly and potentially environmentally
-damaging.
 </p>
-<h1 class="descSub"><i>Oxalobacter formigenes</i> and the
+<h1 class="descSub">Project Overview</h1>
-Breakdown of Oxalate</h1>
 <p class="descP">
-<i>Oxalobacter formigenes</i> is an anaerobic, Gram-
+<b>Step 1: Cloning of <i>frc</i> and <i>oxc</i> into DH5α </b><br>
+-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>
+-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>
+-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>
+-Test ability of enzymes to break down Beerstone<br>
+-Design a functional cleaning solution<br>
-negative bacteria native to the human gut microbiota<span
-class="super">5</span>.  <i>O. formigenes</i> 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 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,
-</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,
-</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>
-<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>
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-Design is the first step in the design-build-test cycle
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-<h3>What should this page contain?</h3>
-<ul>
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Difference between revisions of "Team:UofGuelph/Description"

Revision as of 23:28, 6 December 2018

Project Description

Background on Beerstone, FRC and OXC

Objectives

Project Overview