- 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
-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:
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.
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).