Cloning 7 casein gBlocks, 12Sep2014 and on

Experiment: Cloning 7 casein gBlocks, above, into pD1214, transforming into E. coli, extracting and sequencing plasmid DNA

Overview:

• During this series of experiments, we will insert seven casein gene constructs (detailed below) into the pD1214 vector and transform our seven constructs into E. coli. We will then perform a midiprep plasmid DNA extraction, quantify our plasmid DNA, and submit all seven constructs for sequencing. Sequence-confirmed DNA from this stage is ready to be transformed into yeast.

Casein constructs

1) P.FAKS.bovAlphaS1.S

2) P.FAKS.bovAlphaS2(Kex-).S

3) P.FAKS.bovAlphaS2(Kex+).S

4) P.FAKS.humBeta.S

5) P.FAKS.humKappa(Kex-).S

6) P.FAKS.Beta(A2).S (bov)

7) P.FAKS.bovKappa.S

• These gBlock sequences have 5' and 3' SapI sequences, followed by full length FAKS, followed by human Kappa Casein (Kex+). Electra cloning cleaves at SapI sites and ligates simultaneously.

• We cloned 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.

September 12, 2014: Cloning casein gBlocks into pD1214 & transforming plasmids into E. coli

• Participants: Advait, Patrik, Johan, Minakishi, Rachel, Lafia, Nikola, Aaron, Wes, Arif

• 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.
  • Completed 8 separate reactions, one for each gBlock and one positive control using DNA supplied in the kit.
  • 5uL reaction products used immediate for transformation into E. coli, below. Remainder stored at -20C, BioCurious freezer. Each tube labeled with date and casein construct ID.

Reaction:

1uL pD1214 (20ng)
1uL (20ng) of appropriate casein gBlock or (+) control DNA 
2uL Electra Buffer
1uL of Electra enzyme mix
15uL of Water
----
20uL, Room Temperature, 20min.

• 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 10 reactions: one for each of the 8 Electra cloning reactions, one pGLO positive control, one diH2O 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.
    • Added 5uL of each casein:pD1214 or appropriate control to individual thawed tube of 100uL competent cells. Mixed gently.
    • Incubated immediately on ice for 10 min.
    • Plated 100uL of each reaction to LB carb plates, using one individualy wrapped spreader/reaction.
      • Inducible promoter for pGLO positive control requires arabinose, so 200uL of arabinose spread onto that LB carb plate prior to transformation mix being spread.
    • Incubated plates overnight at 37°C.

September 12, 2014

• Results:
  • All casein:pD1214 --> dH5alpha E. coli transformation reactions yielded colonies on LB carb EXCEPT P.FAKS.humBeta.S. positive and negative control plates as expected.