20.109(S21):M1D3

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20.109(S20): Laboratory Fundamentals of Biological Engineering

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Spring 2020 schedule        FYI        Assignments        Homework        Class data        Communication
       1. Screening ligand binding        2. Measuring gene expression        3. Engineering antibodies              


Introduction

Last time you sorted a population of yeast from a library using FACS based on their ability to bind to lysozyme, our ligand of interest. The cells were selected for having a green fluorescent signal correlated with surface expression of a single-chain antibody fragment (scFv) and a far-red fluorescent signal correlated with binding to lysozyme. The goal of today's experiment is to harvest the plasmid expressing the unique antibody fragment from the mixed population of yeast. You will then transform that mixed population of "improved binder" plasmids from yeast into E. coli bacteria to amplify the plasmid.

Schematic of alkaline lysis: Blue DNA genomic and red DNA plasmid. Image by Qiagen
To harvest plasmid DNA from the yeast cells we will be using a kit that is based off the common E. coli plasmid purification method called alkaline lysis. For yeast there needs to be one additional step to help break down the cell wall, but overall goal of each prep is the same--to separate the plasmid DNA from the chromosomal DNA and cellular debris, allowing the plasmid DNA to be studied further. The key difference between plasmid DNA and chromosomal DNA is size and this difference is what is used to separate the two components.

In the plasmid DNA purification protocol, the media is removed from the cells by centrifugation. The cells are resuspended in an solution that contains Tris to buffer the cells and EDTA to bind divalent cations in the lipid bilayer, thereby weakening the cell envelope. An alkaline (basic) lysis buffer is added that contains sodium hydroxide and the detergent sodium dodecyl sulfate (SDS). The base denatures the cell’s DNA, both chromosomal and plasmid, while the detergent dissolves the cellular proteins and lipids. The pH of the solution is returned to neutral by adding a mixture of acetic acid and potassium acetate. At neutral pH the SDS precipitates from solution, carrying with it the dissolved proteins and lipids. The DNA strands renature at neutral pH. The chromosomal DNA, which is much longer than the plasmid DNA, renatures as a tangle that gets trapped in the SDS precipitate. The smaller plasmid DNA renatures normally and stays in solution, effectively separating plasmid DNA from the chromosomal DNA and the proteins and lipids of the cell. At this point the solution is spun at a high speed and soluble fraction, including the plasmid, is kept for further purification and the insoluble fraction, the macromolecules and chromosomal DNA is pelleted and thrown away.

After isolation and further purification, you will quantify your DNA by spectrophotometry. Nucleic acids (both RNA and DNA) have an absorbance peak at 260 nm. Beer's law may be used to quantify the amount of DNA from this peak: Abs = ε l c, where Abs is the measured absorbance, l is the path length (1 cm for most specs), c is concentration, and ε is the extinction coefficient. For DNA, ε is 0.02 (μg/mL cm)-1, so 1 absorbance unit corresponds to 50 μg/mL of DNA. The absorbance at 280 nm gives some indication of DNA purity, as proteins have their absorbance peaks at that value (primarily due to the aromatic peptides tryptophan and tyrosine). An Abs260:Abs280 ratio of ~1.8:1 is desired.

Finally you will transform the yeast plasmid DNA into E. coli. Yeast are not optimized to create many copies of plasmid DNA and we need a much higher concentration of DNA to analyze the sequence of the plasmid. Luckily for us engineered E. coli are relatively inexpensive and will quickly amplify plasmids. Most E. coli do not usually exist in a state in which they will easily take up plasmid DNA, but the bacteria can be prepared such that they are permeable to the plasmid DNA. The process of taking up foreign DNA into a cell is called transformation, and when cells are prepared to take up DNA they are called competent. The transformation procedure is efficient enough for most lab purposes, with efficiencies as high as 109 transformed cells per microgram of DNA, but it is important to realize that even with high efficiency cells only 1 DNA molecule in about 10,000 is successfully transformed. In our transformation protocol we will adjust the amount of plasmid DNA such that the E. coli will each take up between 0-1 plasmid per competent cell. The cells that do not take up plasmid DNA will not survive in the selective culture media (or on selective media plates). Selective media typically contains an antibiotic which kills microbes that do not produce the necessary enzyme to neutralize the antibiotic. The enzyme for a given antibiotic is encoded in the plasmid DNA. We can then harvest individual colonies of E. coli to find unique sequences of improved binders.

Protocols:

The yeast that were sorted via FACS in our last lab session were incubated at 30°C for an additional three days so the yeast could recover from the sorting and grow. This additional growth time will give us a sufficient number of cells to harvest plasmid DNA.

Part 1: Harvest plasmid DNA from yeast

To harvest plasmid DNA from yeast we need to do a bit more work to break down the cell wall. Yesterday the 2x107 yeast cells were pelleted and digested with zymolase overnight at 37°C. Zymolase is a mixture of enzymes purified from the bacteria Arthrobacter luteus that degrades the cell wall of yeast and other fungi. The yeast plasmid purification will be carried out using reagents from a commercial kit called Zymoprep MiniPrep II.

  1. Retrieve your overnight yeast digests from the 37°C incubator.
  2. Add 200uL of Solution 2 and vortex briefly.
    • This is the alkaline buffer.
  3. Add 400uL of Solution 3, vortex briefly and centrifuge at 14,000g for 10min.
    • This is the buffer that neutralizes the pH.
  4. Transfer the supernatant to a new 1.5 mL tube and centrifuge at 14,000g for an additional 10min.
  5. Transfer the supernatant to a blue DNA binding, silica column and centrifuge at 10,000g for 30sec. Discard the flow through into a tube labeled 'zymo waste'.
  6. Add 550uL DNA wash buffer to the column, centrifuge at 10,000g for 2min, and discard the flow through to 'zymo waste'.
  7. Transfer the column to a new 1.5 mL tube and carefully add 10uL of water to the center of the column to elute the DNA.
  8. Let the column sit for 2min at room temp.
  9. Centrifuge at 10,000g for 1min to elute the plasmid DNA from the column and collect the flow-through.
  10. You will now take your samples to the Nanodrop to measure the DNA's absorbance to quantify the concentration.

Part 2: Transform plasmid from yeast into E. coli

Competent cells are fragile and should be handled gently, specifically kept cold and not vortexed.

  1. Label a 1.5 mL tubes with your team information and chill it in your ice bucket.
  2. Carefully aliquot 25uL of competent NEB 5-alpha E. coli cells from the front laboratory bench in to your cold tube on ice.
  3. Add 5uL of the yeast plasmid DNA to the competent bacteria.
    • Gently tap the tube with your fingertip 5 times to mix.
    • Remember: it is important to keep the competent cells cold. Also, avoid over pipetting and vortexing!
  4. Incubate your transformation mix on ice for 30 min.
  5. Carry your ice bucket with your transformation to the heat block at the front laboratory bench.
    • Be sure you also take your timer.
  6. Transfer the tube with your transformation to the heat block set to 42 °C and incubate for exactly 30 sec.
  7. Remove your transformation from the heat block and immediately put them back in the ice bucket, then incubate for 5 min.
  8. Pipet 500 uL of pre-warmed SOC media into the transformation.
  9. Move your transformation to the 37 °C incubator and carefully place them on the nutator.
  10. Incubate transformation for 1 h.
  11. Retrieve your transformation from the incubator and alert the teaching faculty that you are ready to plate your samples.
  12. Plate 100μL of the transformation onto an appropriately labeled LB+Amp agar plate.
    • The teaching faculty will demonstrate how you should spread the transformation onto the LB+Amp agar plate by using an ethanol burner to sterilize a glass cell spreader.
  13. Move your spread plate to the 37 °C incubator where they will incubate overnight.
Bacterial transformation: Blue cells did not take up plasmid during heat shock and red cells did take up plasmid with antibiotic resistance marker during heatshock.


Part 3: Harvest plasmid (Miniprep) from E. coli

Since the last lab session(Spring 2020~since last part), the transformed E. coli bacteria grew on LB agar plated with ampicillin antibiotic selection overnight at 37°C. Only bacteria that took up plasmid with an ampicillin selection marker during the heat shock were able to grow, the non-transformants died. The next day visible colonies were picked off the LB+amp plates and grown in LB+amp liquid culture overnight. Growing the bacteria in liquid culture allows one to easily harvest plasmid from a high number of cells. Note: The procedure for DNA isolation at this scale (1-2mL) is commonly termed "mini-prep," which distinguishes it from a “maxi-prep” that involves a larger volume (100mL) of cells and additional steps of purification.

  1. Pick up your two cultures, which are growing in test tubes labeled with your team color. Label two 1.5mL tubes to reflect your samples (scFv_clone#1 and #2).
  2. Vortex the bacteria and pour ~1.5 mL of each candidate into a 1.5mL tube.
  3. Balance the tubes in the microfuge, spin them at maximum speed for 2 min, and remove the supernatants with the vacuum aspirator.
    Diagram showing how to aspirate the supernatant. Be careful to remove as few cells as possible.
  4. Resuspend each cell pellet in 250 μL buffer P1.
    • Buffer P1 contains RNase so that we collect only our nucleic acid of interest, DNA.
  5. Add 250 μL of buffer P2 to each tube, and mix by inversion until the suspension is homogeneous. About 4-6 inversions of the tube should suffice. You may incubate here for up to 5 minutes, but not more.
    • Buffer P2 contains sodium hydroxide and SDS for alkaline lysis.
  6. Add 350 μL buffer N3 to each tube, and mix immediately by inversion (4-10 times).
    • Buffer N3 contains acetic acid which neutralizes the pH. The plasmid DNA renatures while chromosomal DNA / proteins / lipids precipitate into visible white aggregates.
  7. Centrifuge for 10 minutes at maximum speed. Note that you will be saving the supernatant after this step.
    • Meanwhile, prepare 2 labeled QIAprep columns, one for each candidate clone. You must add the label directly to the blue column and not the clear tube.
  8. Transfer the entire supernatant to the column and centrifuge for 1 min. Discard the eluant into a tube labeled 'Qiagen waste'.
  9. Add 0.5 mL PB to each column, then spin for 1 min and discard the eluant into the Qiagen waste.
  10. Next wash with 0.75 mL PE, with a 1 min spin step as usual. Discard the ethanol in the Qiagen waste.
  11. After removing the PE, spin the mostly dry column for 1 more minute.
    • It is important to remove all traces of ethanol, as they may interfere with subsequent work with the DNA.
  12. Transfer each column insert (blue) to a 1.5mL tube with the lid cut off.
    • Use the scissors in your drawer to cut the lids off two tubes.
  13. Add 30 μL of distilled H2O pH ~8 to the top center of the column, wait 1 min, and then spin 1 min to elute your DNA.
  14. Cap the trimmed tube or transfer elution to new 1.5mL tube.
  15. You will now take your samples to the Nanodrop to measure the DNA's absorbance to quantify the concentration.

Reagents

  • Zymoprep Yeast Plasmid Miniprep II (from Zymo Research)
    • Zymolyase
    • solution 1
    • solution 2
    • solution 3
    • wash buffer
  • Chemically competent E. coli NEB5a (genotype: fhuA2 Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17)
  • SOC medium: 2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, and 20 mM glucose
  • LB+Amp plates
    • Luria-Bertani (LB) broth: 1% tryptone, 0.5% yeast extract, and 1% NaCl
    • Plates prepared by adding 1.5% agar and 100 μg/mL ampicillin (Amp) to LB
  • QIAprep Spin Miniprep Kit (from Qiagen)
    • buffer P1
    • buffer P2
    • buffer N3
    • buffer PB
    • buffer PE

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