Cloning 7 casein gBlocks, 12Sep2014 and on

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Cloning 7 casein gBlocks 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 is then 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.

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

September 12, 2014

  • Participants: Advait, Patrik, Johan, Meenakshi, Rachel, Lafia, Nikola, Moises, 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.
    • Used DNA 2.0 Electra cloning kit protocol.
    • 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 -20°C, 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.
      • Immediately incubated 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 14, 2014

  • Results:
    • All casein:pD1214 --> dH5alpha E. coli transformation reactions yielded colonies on LB carb EXCEPT P.FAKS.humBeta.S. Few colonies for P.FAKS.humKappa(Kex-).S, and multiple colonies on remaining successful plates,
    • Positive and negative control plates as expected.

Repeating transformation of P.FAKS.humBeta.S:pD1214 into E. coli

September 14, 2014

  • Aims: Of the September 12, 2014 transformations (gBlocks:pD1214 to the dH5alpha strain of E. coli & controls, above), only one failed: P.FAKS.humBeta.S:pD1214. We are repeating this transformation using the Electra cloning reaction from September 12, 2014 into Mix & Go competent E. coli, strain dH5alpha.
  • Participants: Johan, Patrik, Aaron, Meenakshi
  • Location: BioCurious
  • Materials and Methods:
    • Repeated the transformation using Media:Zymo_E._coli_competent_cells_transformation_protocol.pdf‎ as described in Methods September 14, 2014, above, with the following changes:
      • To attempt to improve transformation efficiency, we added an outgrowth step after 10 minute incubation on ice. Incubated transformation reactions in liquid SOC media: 2.5 hours at 37°C.

September 15, 2014

  • Results: No growth for P.FAKS.humBeta.S:pD1214 to dH5alpha E. coli observed on LB carb plates incubated overnight at 37°C.
  • Discussion: Second unsuccessful transformation could have been due to failed 1) transformation 2) Electra cloning or 3) resuspension of IDT gBlock DNA. Ideal to quantify P.FAKS.humBeta.S IDT gBlock resuspension to confirm contains DNA, however, we don't have access to a nanodrop UV spec, and quantifying on BioCurious's Beckman Coulter DU 640 spectrophotometer likely to require more DNA than would be prudent to use (see DNA quantification on September 1, 2014: CLONE 082514 humKappaCasein(Kex+).) Failure of Electra cloning possible due to large number of experimental participants and multiple pipetting steps.
  • Future Directions: We will repeat both Electra cloning and transformation for P.FAKS.humBeta.S.

Plasmid midipreps of successful 12Sep2014 transformations

September 14, 2014

  • Participants: Johan, Patrik, Aaron, Meenakshi
  • Location: BioCurious
  • Aims: Extract the plasmid DNA from the six successful IDT gBlock:pD1214 to dH5alpha transformations (September 12, 2014) using the Zyppy Plasmid Midiprep kit. After quatification, we will submit this DNA for sequencing prior to working with it in yeast.

Casein constructs in pD1214 vector we will be midi-prepping today:

1) P.FAKS.bovAlphaS1.S

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

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

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

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

7) P.FAKS.bovKappa.S

  • Materials and Methods:
    • We used the Zyppy Plasmid Midiprep Kit (Protocol = Media:D4025i.pdf‎):
      • Followed "Centrifugation Protocol" stream with the following specific changes to step 1): grew in 12mL of LB Amp and added 6mL of water to the bacterial cell pellet.
    • Midiprepped DNA is stored at -20°C, in the freezer at BioCurious.

Repeating Electra cloning and transformation for P.FAKS.humBeta.S

September 15, 2014

Participants: Nikola, Johan, Meenakshi, Lafia, Wes, Richard, Matt

Location: BioCurious

Aims: As we had two previous unsuccessful transformations (12Sep1014, 14Sep2014) using P.FAKS.humBeta.S:pD1214 cloning reaction from 12Sep2014, we are going to repeat the Electra cloning reaction for this strain, then repeat the transformation.

Materials & Methods:

* Materials & Methods: cloning P.FAKS.humBeta.S into pD1214

Reaction: 

1uL pD1214 (20ng)
1uL (20ng) P.FAKS.humBeta.S or (+) control DNA from Electra cloning kit
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 4 reactions: P.FAKS.humBeta.S:pD1214, one Electra cloning positive control, one pGLO positive control, one 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.
      • Added 5uL of each casein:pD1214 or appropriate control to individual thawed tube of 100uL competent cells. Mixed gently.
      • Immediately incubated 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.
  • Results & Discussion:
    • This transformation reaction yielded colonies, so likely previous failed transformations due to unsuccessful Electra cloning reaction of P.FAKS.humBeta.S on 12Sep2014. We will choose two colonies for plasmid DNA extraction.

Plasmid DNA quantification & purity spec readings for 14Sep2014 midipreps

September 16,2014

  • Participants: Aaron, Meenakshi, Johan, Lafia, Matt
  • Location: BioCurious
  • Aims: DNA quantification of all midipreped sample by using spectrophotometer.
  • Materials and Methods:

We used 1:10 dilution. Sample no 3 ( P. FAKS. Bovine alpha ( Kex +).s had shown less concentration because in 1 tube from duplicate set had less growth during midiprep.

Sample. 260nm. 280nm. 260/280. micro g / ml

1. 0.0748. 0.0043. 1.8546. 3.7416

2. 0.0412. 0.0240. 1.7177. 2.0616

3. 0.0174. 0.0095. 1.8264. 0.08688

5. 0.0479. 0.0256. 1.8697. 2.3935

6. 0.0316. 0.0148. 2.1362. 1.5819

7. 0.0693. 0.0342. 2.0245. 3.4632

Midiprep of P.FAKS.humBeta.S:pD1214 from dH5alpha E. coli

September 17, 2014

  • Participants: Patrik, Meenakshi, Lafia, Nikola, Johan
  • Location: BioCurious
  • Aims: Midiprep TWO colonies of repeat P.FAKS.humBeta.S:pD1214 in dH5alpha. Selecting two colonies as this gene insert has been difficult in the past.
  • Materials and Methods:
    • We used the Zyppy Plasmid Midiprep Kit (Protocol = Media:D4025i.pdf‎):
      • Followed "Centrifugation Protocol" stream with the following specific changes to step 1):
        • In morning, inoculated two colonies from transformation September 15, 2014 each into 12 mL LB Amp liquid media. Incubated in 37°C shaking incubator until 7pm.
        • Added 6mL of water to the bacterial cell pellet.
    • Midiprepped DNA is stored at -20°C in the BioCurious freezer.
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