Difference between revisions of "Team:UofGuelph/Description"

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<h1 class="descHead">THIS IS THE 2017 TEMPLATE PAGE, NEED TO UPDATE</h1>
 
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<h1 class="descHead">Project Description</h1>
 
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<h1 class="descHead">Background on Beerstone, FRC, OXC and OxIT</h1>
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<h1 class="descSub">Background on Beerstone, FRC and OXC</h1>
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<h1 class="descSub">Beerstone, FRC and OXC</h1>
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Calcium oxalate build up (commonly referred to as Beerstone) is a major ongoing problem in the brewing industry. Beerstone is difficult to remove from brewing equipment, requiring the use of harsh cleaning chemicals, and provides a porous, nutrient rich growing environment for bacterial biofilms. Our project is to develop a biological, enzyme based cleaning method for the removal of Beerstone from brewing equipment. We propose to use the enzymes FRC and OXC from <i> Oxalobacter formigenes </i> oxalate degrading metabolic pathway to breakdown Beerstone. </p>
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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>.
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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>.
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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>.
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<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>.
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<h1 class="descSub">Project Overview</h1>
 
<h1 class="descSub">Project Overview</h1>

Revision as of 00:22, 18 October 2018

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THIS IS THE 2017 TEMPLATE PAGE, NEED TO UPDATE

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 beerstone2,3. 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.

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

The 2017 Aspect

Due to setbacks and unforeseen circumstances our team was only able to accomplish Step 1 this year. We will however be continuing our project next year where we left off. This winter we will be re-grouping in an effort to better plan for next year so that our project execution occurs more smoothly and we are able to accomplish more.

University of Guelph iGEM 2017