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 B

9) Sap.Faks.hFam20C(Kex+).Sap = this is the gene for the FAM20C kinase involved in phosphorylating secreted proteins, including caseins. The DNA sequence of our "Kex+" variant translates to 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.

Cloning casein & FAM20C gBlocks into pD1214 & transforming plasmids into E. coli

September 27, 2014

• Participants: Maria, Gabby, Rebecca, Johan, Meenakshi, Rachel, Lafia, Nikola

• Location: BioCurious

• Materials & Methods, Electra Cloning:
  • We resuspended all lyophilized IDT gBlock DNA as in DNA Handling using 0.5X TE (1X TE, pH 7.5: 1 diH2O). Final DNA concentration = 20ng/uL, per Electra cloning kit requirements.
  • Used DNA 2.0 Electra cloning kit protocol.

Reaction:

1uL pD1214 (20ng)
1uL of appropriate casein gBlock or (+) control DNA (20ng), or 0.5X TE (-) control
2uL Electra Buffer
1uL of Electra enzyme mix
15uL of diH20
----
20uL

• • As possible that multiple pipetting stages contributed to failed cloning reaction for P.FAKS.humBeta.S 12Sep2014 (Cloning 7 casein gBlocks, 12Sep2014 and on), we're making a master mix that includes all reagents common to each reaction. Adding 5.5X each reagent for 5 reactions and 0.5 extra.

Master mix ( MUST BE MIXED THOROUGHLY ) and cloning reactions:

5.5uL pD1214 (20ng/uL)
11.0uL Electra Buffer
5.5uL of Electra enzyme mix
82.5uL of diH20
----
19uL of master mix to each reaction tube + 1uL appropriate gBlock DNA (20ng/uL) or control. 
20 min at room temperature.

• • Completed 5 separate reactions, one for each gBlock, one positive control using DNA supplied in the kit, one negative control with 0.5XTE resuspension buffer in place of DNA insert.
  • 5uL reaction products used immediate for transformation into E. coli, below. Remainder stored at -20°C, BioCurious freezer. Each tube labeled with date and construct ID.

• Materials & Methods, Transforming Mix & Go Competent dH5alpha E. coli with gBlocks in pD1214, above:
  • We used the Zymo Research instruction manual for "Premade Mix & Go Competent E. coli Cells." Media:Zymo_E._coli_competent_cells_transformation_protocol.pdf‎
  • We completed 7 reactions: one for each of the 5 Electra cloning reactions, one pGLO positive control, one competent cells only negative control.
    • Thawed individual tubes of Mix & Go chemically competent, dH5alpha E. coli cells slowly on ice.
    • Prewarmed LB agar plates with Carb-50, 100mm (Teknova), at 37°C for until temperature read >34°C with temperature scanner: 1 hr 10 min.
      • Inducible promoter for pGLO positive control requires arabinose, so used a plate that already contained [need concentration] of arabinose for that reaction.
    • Added 5uL of each casein:pD1214 or appropriate control to individual thawed tube of 100uL competent cells. Mixed gently.
    • Immediately incubated on ice for 30 min.
    • In UV & 70% isopropanol-sterilized laminar flow hood, plated 105uL of each reaction to LB carb plates, using one individually wrapped spreader/reaction.
    • Incubated upside down plates overnight at 37°C.

September 28, 2014

• Results
  • The three experimental plates (Bovine Beta B, FAM20C Kex +, FAM20C Kex- in pD1214), Electra cloning positive control and PGlo transformation positive control had >1000 colonies each.
  • The transformation negative control plate had no colonies.
  • However, the Electra cloning negative control (control that included all Electra cloning reagents and the TE buffer in which we resuspended our IDT gBlocks; excluding only IDT gBlock "mother vector" DNA), had 46 colonies. Problem!

• Discussion and Future Directions
  • Troubleshooting failed negative control: As transformation negative control plate was blank, contamination unlikely to have occurred while plating in flow hood or in competent cells. Colonies also unlikely due to non-functional antibiotics in LB plates. Some possibilities: 1) contamination of Electra reagents or protocol equipment (pipettes, etc.) by plasmid conferring amp-resistance (one of our plasmids or another AmpR plasmid somehow ambient in lab) or 2) contamination of same by AmpR E. coli cells. 3) Perhaps the linearized pD1214 plasmid can self-ligate at some low frequency, despite incompatible overhangs.
    • We will plate 1uL of each Electra reagent and the 0.5X TE resuspension buffer (27Sep2014 cloning, above) brought to 100uL in our diH20 on an LB carb plate to see if resistant E. coli colonies grow.
    • We will perform a plasmid midiprep DNA extraction on a liquid culture of one of the colonies from the Electra negative control plate for sequencing and potential further characterization. This will allow us to tell whether this colony contains self-ligated pD1214 or one of our insert-containing plasmids.
  • As the experimental plates grew so many more colonies than the failed Electra negative control, many of the colonies likely grew from a cell containing our target plasmid. We will also inoculate liquid cultures from 3 colonies per plate for plasmid DNA extraction and DNA sequencing.