Cloning strategy

Milestone

Our first milestone will be to express and secrete full-length kappa-casein in S. cerevisiae. We only get one discounted order of gBlocks from IDT so we will save that for later milestones and pay full price for the initial kappa-casein synthesis.

Once the first milestone has been accomplished, we will attempt express

Orders for first milestone

E. coli

• E. cloni 10G: We are requesting a donation from NEB

S. cerevisiae

• The strain must be a URA3 knockout: We are requesting a donation from NEB

Shuttle vector

The shuttle vector is a vector that can be grown and selected in E. coli and S. cerevisiae, but which only correctly expresses the gene or genes of interest in S. cerevisiae.

We're ordering the pD1214-FAKS shuttle vector from DNA 2.0 which contains the following features:

• AmpR: Ampicillin resistance gene (for selection in E. coli)
• P-Amp: Constitutive promoter for AmpR
• pUCori: High copy number E. coli origin of replication
• 2um: High copy number S. cerevisiae origin of replication from 2-μm plasmid.
• URA3: Yeast selectable marker. Requires the use of an S. cerevisiae strain missing the URA3 gene.
• P_ura: Constitutive S. cerevisiae promoter for URA3
• T_ura: S. cerevisiae terminator for URA3
• P_TEF1_syn: Constitutive S. cerevisiae promoter for our gene of interest (we will swap this out for an inducible promoter later)
• Secretion signal: Full alpha-factor yeast secretion signal. Fused to our gene of interest to secrete the protein.
• CYC1terminator: S. cerevisiae terminator for our gene of interest.

The shuttle vector will arrive linearized with overlaps for DNA 2.0's own Electra cloning system.

Reagents

• High fidelity PCR reagents. The shuttle vector will have to be PCR'ed to remove the constitutive promoter and add the appropriate sites for our cloning strategy of choice.

Craig is working on a full reagent list.

Orders for second milestone

This is a work in progress. So far we're planning a large order of gBlocks from IDT including at least some of the following sequences:

Bovine:

• Full-length bovine κ Casein
  • B genetic variant AAQ87923.1
• Full-length bovine αs1 Casein
• Full-length bovine αs2 Casein
• Full-length bovine β Casein
  • A2 genetic variant for health benefit
  • B genetic variant for better coagulation

Human:

• Full-length human κ Casein P07498.3
• Full-length human αs1 Casein
• Full-length human αs2a Casein CSN1S2A
• Full-length human αs2b Casein CSN1S2B
• Full-length human β Casein
• human caseinomacropeptide
• FAM20C Kinase

Whale:

We don't have the sequences yet!