Difference between revisions of "Team:UofGuelph/Description"
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| − | + | - 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. |
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| − | <h1 class="descSub"> | + | <h1 class="descSub">Project Overview</h1> |
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| + | <b>Step 1: Cloning of <i>frc</i> and <i>oxc</i> into DH5α </b> | ||
| + | -Synthesize <i>frc</i> and <i>oxc</i> | ||
| + | -Add PstI cut site to pET-28a | ||
| + | -Ligate <i>frc</i> and <i>oxc</i> into pET-28a | ||
| + | -Transform pET-28afrc/oxc into DH5α | ||
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| + | <b>Step 2: Clone <i>frc</i> and <i>oxc</i> into BL21</b> | ||
| + | -Purify pET-28afrc/oxc from DH5α | ||
| + | -Transform pET-28afrc/oxc into BL21 | ||
| + | |||
| + | <b>Step 3: Express and Purify FRC and OXC</b> | ||
| + | -Induce expression with IPTG and extract crude proteins | ||
| + | -Purify proteins using Ni-NTA chromatography | ||
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| + | <b>Step 4: Characterize FRC and OXC</b> | ||
| + | -Characterize enzyme function using Sodium Oxalate | ||
| + | -Characterize enzyme function using Calcium Oxalate | ||
| + | |||
| + | <b>Step 5: Design a Cleaning Solution and Test on Beerstone</b> | ||
| + | -Test ability of enzymes to break down Beerstone | ||
| + | -Design a functional cleaning solution | ||
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Revision as of 02:49, 18 October 2018
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.
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
