Difference between revisions of "Team:UofGuelph/Design"

 
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<h1 class="descHead">Project Design</h1>
 
<h1 class="descHead">Project Design</h1>
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<img src="https://static.igem.org/mediawiki/2017/4/4a/T--U_of_Guelph--gryphon.jpg" class="guelphImages">
+
<img src="https://static.igem.org/mediawiki/2017/4/4a/T--
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<h1 class="descSub">Whats the Deal with Beerstone?</h1>
+
 
 +
U_of_Guelph--gryphon.jpg" class="guelphImages">
 +
 
 +
 
 +
<h1 class="descSub">Immediate Objectives</h1>
 
<p class="descP">
 
<p class="descP">
−
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 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>
+
- Express FRC and OXC in <i>E. coli</i> BL21 using pET28a
−
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. 
+
vector.
 +
- Assess the feasibility of using these enzymes as an  
 +
 
 +
alternate cleaning method to degrade beerstone.
 
</p>
 
</p>
  
−
<h1 class="descSub"><i>Oxalobacter formigenes</i> and the Breakdown of Oxalate</h1>
+
<h1 class="descSub">Project Overview</h1>
 +
 
 
<p class="descP">
 
<p class="descP">
−
<i>Oxalobacter formigenes</i> is an anaerobic, Gram-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>.
+
<b>Step 1: Cloning of <i>frc</i> and <i>oxc</i> into DH5α
−
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>:
+
 
 +
</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>
 +
 
 +
</p>
 +
 
 +
<h1 class="descSub">Yeast Project Expansion</h1>
 +
 
 +
<p class="descP">
 +
<i>Saccharomyces cerevisiae</i> is a well characterized expression system for heterologous proteins<sup>1</sup>. iGEM Guelph proposed the use of an isogenic wildtype <i>S. cerevisiae</i> strain, (W303α) along with two expression systems:
 +
<br>
 +
1. pD1218 that would have <i>frc</i>, <i>oxc</i> and <i>oxit</i> inserted into the plasmid that will allow for the transformed strain (<i>S. cerevisiae-Ox</i>) to be able to endogenously breakdown oxalate.
 +
<br>
 +
2. pD1218-Full α-MF-<i>frc</i>/<i>oxc</i> that would have two separate plasmids transformed into two separate yeast to heterologously express <i>frc</i> and <i>oxc</i> simultaneously that can then be purified for further characterization. 
 
<br><br>
 
<br><br>
 +
pD1218 was used because it is an episomal plasmid that contained<sup>2</sup>:
 +
<br>
 +
1. 2μm, an origin of replication so the cell can maintain a high copy number of pD1218-<i>frc</i>/<i>oxc</i>/<i>oxit</i>.
 +
<br>
 +
2. TEF1, a constitutive promoter that has strong promoter activity in yeasts.
 +
<br>
 +
3. Geneticin-r, resistance to the antibiotic G418 that is the selection marker for yeasts.
 +
<br>
 +
4. pUC, an <i>E. coli</i> ori that allows for immense copies of pD1218 when inserted in <i>E. coli</i>.
 +
<br>
 +
5. Ampicillin-r, <i>E. coli</i> transformed with pD1218 will have resistance to the β-lactam.
 +
<br>
 +
6. CYC1, a 3’ UTR that controls post transcriptional regulation
 +
<br><br><br>
 +
 +
 +
<b>Project 1 - Creating and characterizing <i>S. cerevisiae</i>-Ox</b>
 +
<br><br>
 +
pD1218-<i>frc</i>-<i>oxc</i>-<i>oxit</i> will be made using cloning and this it does not possess any genetic elements that will allow the yeast to endogenously secrete plectasin. Instead, we hope to provide the genetic circuitry to enable it to have the biosynthetic metabolism to break down oxalate <i>in situ</i>.
 +
<br>
 +
After cloning in <i>frc</i>, <i>oxc</i> and <i>oxit</i> into W303α, our developed system will have its biological parameters defined. Important questions to address will be:
 +
<br>
 +
1. At what concentration of oxalate will growth of <i>S. cerevisiae</i>-Ox be inhibited?
 +
<br>
 +
2. What’s the efficiency of the rate of oxalate breakdown by <i>S. cerevisiae</i>-Ox at different sub-inhibitory concentrations?
 +
<br>
 +
Answers can be found using an adapted Minimum Inhibitory Concentration (MIC) assay. It will be carried out to CLSI established guidelines where an initial concentration of oxalate will be dissolved into YPD media and then serially diluted in a 96-well plate. Culture of  <i>S. cerevisiae</i>-Ox will then be diluted to an OD of 0.2 and added to the oxalate-rich media.
 +
<br>
 +
At 24, 48 and 72 hour time points, the OD values will be recorded at each concentration to give values that provide information on the effects of oxalate presence of growth of <i>S. cerevisiae-Ox</i>. An oxalate dissolution test kit from Trinity BioLabs can then be used to measure the rate at which different concentrations of oxalate are broken down by <i>S. cerevisiae</i>-Ox.
 +
<br><br><br>
 +
 +
 +
 +
<b>Project 2 - The heterologous expression and protein characterization of <i>frc</i> and <i>oxc</i>.</b>
 +
<br><br>
 +
To ensure the secretion of FRC and OXC proteins to extract and measure different expression conditions, further pD1218-based plasmids were developed. These plasmids contained a full α-mating factor (α-MF) leader peptide for secretion with modified Hexahistadine (H6) tags to allow for protein detection using antibody probing.
 +
<br>
 +
The additional components used were:
 +
<br>
 +
1. Full α-MF, an 89aa secretion propeptide from the yeast α mating factor that has protease cleavage sites to naturally cleave off the α-MF protein sequence during protein trafficking.This will allow for secretion of an unmodified form of the protein of interest (POI), plectasin.
 +
<br>
 +
2. α-F Base, a secretion-signal peptide that is naturally cleaved after it aids in translocating the plectasin, to the cell surface.
 +
<br>
 +
3. GH6A, a glycine-hexahistidine-alanine tag that was modified to mask the positive charge of the H6-tag to avoid any electrostatic interactions between H6, plectasin or the cytoplasmic membrane.
 +
<br><br>
 +
 +
 +
In total 2 unique pD1218-based plasmids will be created:
 +
<br>
 +
1. pD1218-full-α-MF-GH6A-<i>frc</i>
 +
<br>
 +
2. pD1218-full-α-MF-GH6A-<i>oxc</i>
 +
<br><br>
 +
 +
These plasmids would be transformed and amplified in strains of <i>E. coli</i> DH5α to obtain large amounts of pDNA so that it could have been transformed into the <i>S. cerevisiae</i> strain, W303α.
 +
<br>
 +
The secreted FRC and OXC will be purified using a Ni-NTA column and then characterized by microbroth confrontation assays to assess the optimal ratios needed for efficient breakdown of oxalate.
 +
<br><br><br>
 +
 +
 +
 +
 
</p>
 
</p>
  
−
<h1 class="descSub">Our Project Design</h1>
+
<h1 class="descSub">References</h1>
 +
<p class="descP">
  
−
<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;
 
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For who would bear the whips and scorns of time,
 
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The oppressor’s wrong, the proud man’s contumely,
 
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The pangs of despised love, the law’s delay,
 
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The insolence of office and the spurns
 
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That patient merit of the unworthy takes,
 
−
When he himself might his quietus make
 
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With a bare bodkin? who would fardels bear,
 
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To grunt and sweat under a weary life,
 
−
But that the dread of something after death,
 
−
The undiscover’d country from whose bourn
 
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No traveller returns, puzzles the will
 
−
And makes us rather bear those ills we have
 
−
Than fly to others that we know not of?
 
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Thus conscience does make cowards of us all;
 
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And thus the native hue of resolution
 
−
Is sicklied o’er with the pale cast of thought,
 
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And enterprises of great pith and moment
 
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With this regard their currents turn awry,
 
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And lose the name of action.–Soft you now!
 
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The fair Ophelia! Nymph, in thy orisons
 
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Be all my sins remember’d.</p>
 
  
−
<h1 class="descSub">Future Development</h1>
+
1. Thukral, S. K., Chang, K. K. H. & Bitter, G. A. Functional Expression of Heterologous Proteins in Saccharomyces cerevisiae. <i>Methods</i> <b>5</b>, 86–95 (1993).
−
<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:
+
<br>
−
Whether ’tis nobler in the mind to suffer
+
2. Chan, K.-M., Liu, Y.-T., Ma, C.-H., Jayaram, M. & Sau, S. The 2 micron plasmid of Saccharomyces cerevisiae: A miniaturized selfish genome with optimized functional competence. <i>Plasmid</i> <b>70</b>, 2–17 (2013).
−
The slings and arrows of outrageous fortune,
+
</p>
−
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">Refrences</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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<div class="column full_size">
 
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<h1>Design</h1>
 
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<p>
 
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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.
 
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</p>
 
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</div>
 
−
 
−
 
−
 
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<div class="column two_thirds_size">
 
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<h3>What should this page contain?</h3>
 
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<ul>
 
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<li>Explanation of the engineering principles your team used in your design</li>
 
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<li>Discussion of the design iterations your team went through</li>
 
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<li>Experimental plan to test your designs</li>
 
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</ul>
 
−
 
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</div>
 
  
  

Latest revision as of 01:39, 8 December 2018

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Project Design

Immediate 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

Yeast Project Expansion

Saccharomyces cerevisiae is a well characterized expression system for heterologous proteins1. iGEM Guelph proposed the use of an isogenic wildtype S. cerevisiae strain, (W303α) along with two expression systems:
1. pD1218 that would have frc, oxc and oxit inserted into the plasmid that will allow for the transformed strain (S. cerevisiae-Ox) to be able to endogenously breakdown oxalate.
2. pD1218-Full α-MF-frc/oxc that would have two separate plasmids transformed into two separate yeast to heterologously express frc and oxc simultaneously that can then be purified for further characterization.

pD1218 was used because it is an episomal plasmid that contained2:
1. 2μm, an origin of replication so the cell can maintain a high copy number of pD1218-frc/oxc/oxit.
2. TEF1, a constitutive promoter that has strong promoter activity in yeasts.
3. Geneticin-r, resistance to the antibiotic G418 that is the selection marker for yeasts.
4. pUC, an E. coli ori that allows for immense copies of pD1218 when inserted in E. coli.
5. Ampicillin-r, E. coli transformed with pD1218 will have resistance to the β-lactam.
6. CYC1, a 3’ UTR that controls post transcriptional regulation


Project 1 - Creating and characterizing S. cerevisiae-Ox

pD1218-frc-oxc-oxit will be made using cloning and this it does not possess any genetic elements that will allow the yeast to endogenously secrete plectasin. Instead, we hope to provide the genetic circuitry to enable it to have the biosynthetic metabolism to break down oxalate in situ.
After cloning in frc, oxc and oxit into W303α, our developed system will have its biological parameters defined. Important questions to address will be:
1. At what concentration of oxalate will growth of S. cerevisiae-Ox be inhibited?
2. What’s the efficiency of the rate of oxalate breakdown by S. cerevisiae-Ox at different sub-inhibitory concentrations?
Answers can be found using an adapted Minimum Inhibitory Concentration (MIC) assay. It will be carried out to CLSI established guidelines where an initial concentration of oxalate will be dissolved into YPD media and then serially diluted in a 96-well plate. Culture of S. cerevisiae-Ox will then be diluted to an OD of 0.2 and added to the oxalate-rich media.
At 24, 48 and 72 hour time points, the OD values will be recorded at each concentration to give values that provide information on the effects of oxalate presence of growth of S. cerevisiae-Ox. An oxalate dissolution test kit from Trinity BioLabs can then be used to measure the rate at which different concentrations of oxalate are broken down by S. cerevisiae-Ox.


Project 2 - The heterologous expression and protein characterization of frc and oxc.

To ensure the secretion of FRC and OXC proteins to extract and measure different expression conditions, further pD1218-based plasmids were developed. These plasmids contained a full α-mating factor (α-MF) leader peptide for secretion with modified Hexahistadine (H6) tags to allow for protein detection using antibody probing.
The additional components used were:
1. Full α-MF, an 89aa secretion propeptide from the yeast α mating factor that has protease cleavage sites to naturally cleave off the α-MF protein sequence during protein trafficking.This will allow for secretion of an unmodified form of the protein of interest (POI), plectasin.
2. α-F Base, a secretion-signal peptide that is naturally cleaved after it aids in translocating the plectasin, to the cell surface.
3. GH6A, a glycine-hexahistidine-alanine tag that was modified to mask the positive charge of the H6-tag to avoid any electrostatic interactions between H6, plectasin or the cytoplasmic membrane.

In total 2 unique pD1218-based plasmids will be created:
1. pD1218-full-α-MF-GH6A-frc
2. pD1218-full-α-MF-GH6A-oxc

These plasmids would be transformed and amplified in strains of E. coli DH5α to obtain large amounts of pDNA so that it could have been transformed into the S. cerevisiae strain, W303α.
The secreted FRC and OXC will be purified using a Ni-NTA column and then characterized by microbroth confrontation assays to assess the optimal ratios needed for efficient breakdown of oxalate.


References

1. Thukral, S. K., Chang, K. K. H. & Bitter, G. A. Functional Expression of Heterologous Proteins in Saccharomyces cerevisiae. Methods 5, 86–95 (1993).
2. Chan, K.-M., Liu, Y.-T., Ma, C.-H., Jayaram, M. & Sau, S. The 2 micron plasmid of Saccharomyces cerevisiae: A miniaturized selfish genome with optimized functional competence. Plasmid 70, 2–17 (2013).

University of Guelph iGEM 2018