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. coli NEB-10: We have this strain available in our freezer.

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

Since we are using the DNA2.0 Elektra System to clone full-length Kappa-Casein, our reagents list is significantly shorter! Here it is:

Cloning Elektra Vector (Craig Donated) Elektra System (Craig Donated)

E. coli Cloning Carbenicillin (50mg/mL, 25mL) donated by Teknova LB Broth (1L) donated by Teknova LB Carb 50Plates (20) donated by Teknova NEB 10-beta Competent E. coli (High Efficiency)

Genes SapI.KappaCasein.SapI (human) (Craig Donated)

Saccharomyces Expression Saccharomyces cerevisae (Ura3ko)? UV-sensitive, other muts LiOAC (250kg) PEG (500mL, 50%) YPD Plates + uridine (Ura3- growth) YPD Plates (Ura3+ selection; animal free) YPD Broth + Uridine (80ug/mL; 25 tubes/rack) YPD Broth (animal Free Soytone, 1000mL) donated by Teknova

PPE Sterile Nitrile gloves (50/box) Sterile Nitrile gloves (50/box) Biohazard Bags

Consumables 20uL Pipette Tips 200uL Pipette tips 1000uL Tips 1.5mL Eppendorf PCR tubes Plasmid miniPrep

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
  • C genetic variant ACG63494.1
• Full-length bovine αs2 Casein
  • A variant P02663
• Full-length bovine β Casein
  • A2 genetic variant for health benefit P02666.2
  • B genetic variant for better coagulation AAI11173.1

Human:

• Full-length human κ Casein P07498.3
• Full-length human αs1 Casein
  • P47710.1
• (There is no human αs2a Casein, just two truncated pseudogenes)
• Full-length human β Casein
  • P05814.4
• human caseinomacropeptide (why?)
• FAM20C Kinase
  • Q8IXL6

Whale:

We don't have the sequences yet!

Experiments

• DNA Handling
• CR_062214_humanKappaCasein_001
• CLONE_082514_humKappaCasein(Kex+)

Progress summary, Milestones 1 & 2

gBlock gene construct	Ordered, IDT	Received, IDT	Cloned into pD1214 shuttle vector	Plasmid construct transformed into E. coli	Plasmid prep	Construct sequence confirmed
P.FAKS.bovAlphaS1.S	Aug. 18, 2014	Sep. 2, 2014	Sep. 12, 2014	Sep. 12, 2014	Sep. 14, 2014 Ambig. seq. Resubmit and/or reprep new clone/s.
P.FAKS.bovAlphaS2(Kex+).S	Aug. 18, 2014	Sep. 2, 2014	Sep. 12, 2014	Sep. 12, 2014	Sep. 14, 2014 Wrong seq. Reprep new clone/s.
P.FAKS.bovAlphaS2(Kex-).S	Aug. 18, 2014	Sep. 1, 2014	Sep. 12, 2014	Sep. 12, 2014	Sep. 14, 2014	Oct. 1, 2014
P.FAKS.Beta(A2).S (bov)	Aug. 18, 2014	Sep. 2, 2014	Sep. 12, 2014	Sep. 12, 2014	Sep. 14, 2014 Ambig. seq. Resubmit and/or reprep new clone/s.
P.FAKS.bovBeta(B).S	Aug. 18, 2014	Sep. 16, 2014	Sep. 27, 2014	Sep. 27, 2014	Sep. 29, 2014	Oct. 1, 2014
P.FAKS.bovKappa.S	Aug. 18, 2014	Sep. 2, 2014	Sep. 12, 2014	Sep. 12, 2014	Sep. 14, 2014	Oct. 1, 2014
P.FAKS.humAlphaS1.S	Aug. 18, 2014 Redesigned & ordered Sep. 26, 2014	Oct. 3, 2014 (Sep. 26 order) IDT synth. FAIL Sep. 4, 2014
P.FAKS.humBeta.S	Aug. 18, 2014	Sep. 2, 2014	Sep. 12, 2014 Sep. 15, 2014 = Rxn repeat	Sep. 12, 2014 = Rxn fail Sep. 14, 2014 = Rxn fail Sep. 15, 2014 = + colonies	Sep. 17, 2014 prep of Sep. 15 tfm	Oct. 1, 2014
P.FAKS.humKappa(Kex+).S	Aug. 18, 2014	Aug. 25, 2014	Aug. 25, 2014	Aug. 26, 2014	Aug. 27, 2014	Sep. 6, 2014
P.FAKS.humKappa(Kex-).S	Aug. 18, 2014	Sep. 1, 2014	Sep. 12, 2014	Sep. 12, 2014	Sep. 14, 2014 Wrong seq. Reprep new clone/s.
Sap.FAKS.hFam20C(Kex+).Sap	Aug. 18, 2014	Sep. 16, 2014	Sep. 27, 2014	Sep. 27, 2014	Sep. 29, 2014
Sap.Faks.hFam20C(Kex-).Sap	Aug. 18, 2014	Sep. 24, 2014	Sep. 27, 2014	Sep. 27, 2014	Sep. 29, 2014