Difference between revisions of "Team:UofGuelph/Design"
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Revision as of 23:34, 6 December 2018
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.
- 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.
Step 1: Cloning of frc and oxc into DH5α
Project Design
Background on Beerstone, FRC and
OXC
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
Project Overview
-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
