Cloning bovine beta casein, Fam20C (Kex + & -), from 27Sep2014
Cloning bovine beta casein and Fam20C Kex +/- gBlocks into pD1214, transforming into E. coli, extracting and sequencing plasmid DNA
Overview
During this series of experiments, we will insert three gene constructs, below, into the pD1214 vector and transform our constructs into E. coli. We will then perform midiprep plasmid DNA extractions, quantify our plasmid DNA, and submit all three plasmid constructs for sequencing. Sequence-confirmed DNA is then ready to be transformed into yeast.
Genes to be cloned & pD1214 vector
8) P.FAKS.bovBeta(B).S = bovine beta casein
9) Sap.Faks.hFam20C(Kex+).Sap = this is the gene for the FAM20C kinase involved in phosphorylating secreted proteins, including caseins. Our "Kex+" variant is the native amino acid sequence, including the recognition sequences for the Kex protease.
10) Sap.Faks.hFam20C(Kex-).Sap = this is the gene for the FAM20C kinase involved in phosphorylating secreted proteins, including caseins. In case the Kex protease cleaves this protein before activity, our "Kex-" variant eliminates amino acid recognition sequences for the Kex protease by replacing with biochemically similar amino acids.
- These Integrated DNA Technologies (IDT) gBlock sequences have 5' and 3' SapI sequences, followed by full length FAKS, followed by the gene of interest, codon-optimized for expression in S. cerevisiae. Electra cloning cleaves at SapI sites and ligates simultaneously.
- We will clone into pD1214. This vector was ordered from DNA2.0 and is linearized upon arrival. Our insert is treated with Electra enzyme mix which cuts and ligates our "SapI.gene.SapI" and inserts it into the pD1214 vector.
- SapI treated DNA has an ATG overhang at the 5' end and a GGT overhang at the 3' end. Our complete vector will have the alphafactorfull signal sequence, which leads into another Methionine (M), followed by the rest of the human kappa casein protein.
All you need to know about the Electra system.