20.109(F21):M2D3

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20.109(F21): Laboratory Fundamentals of Biological Engineering
Drawing provided by Marissa A., 20.109 student in Sp21 term.  Schematic generated using BioRender.

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       Module 1: Genomic instability                          Module 2: Drug discovery       


Introduction

To induce production of PF3D7_20109-F21 protein from the expression plasmid that was 'cloned' in the previous laboratory session, a lactose-analogue isopropyl β-D-1-thiogalactopyranoside (IPTG) was used to induce expression in Nico(DE3) E. coli bacterial cells. The use of IPTG to induce protein expression is based on the native lac operon used for lactose metabolism in bacterial cells.

Sp17 20.109 M1D2 lac operon.png
Sp17 20.109 M1D2 lactose vs IPTG.png
The lac operon is composed of four genes: lacI, lacZ, lacY, and lacA. When lactose is absent, LacI (the protein encoded by lacI) binds to the operator sequence (O) upstream of lacZYA. In the presence of lactose, LacI and lactose form a complex which relieves repression of lacZYA transcription. LacZ is a β-galactosidase that cleaves lactose resulting in glucose and galactose. LacY, a β-galactoside permease, facilitates the transport of lactose across the cell membrane, and LacA, a β-galactoside transacetylase, transfers an acetyl group from acetyl-CoA to β-galactosides.

The native lac operon is a powerful tool in engineering protein expression systems because it enables researchers to control gene expression using inducer molecules. The lacZYA genes are only expressed when lactose is present. If a gene of interest is cloned downstream of the operator sequence, the expression of this gene can be controlled by LacI repression and lactose derepression. To further control the system for protein expression, IPTG is used as a lactose-analog as it is not metabolized by the cells.

Today you will isolate the expressed PF3D7_20109-F21 protein from the bacterial cells. Remember that the PF3D7_20109-F21 gene sequence was synthesized using a gBlock. When the gBlock was cloned into the expression vector, a Strep-Tactin was incorporated into to the DNA sequence. The resultant protein is therefore Strep-Tactin-tagged. Strep-Tactin is a variant of streptavidin is a 66 kDa protein from the bacterium Streptomyces avidinii. In molecular biology streptavidin is a useful tool due to having a very high affinity for biotin with a dissociation constant (Kd) of ~10-14 mol/L, which is one of the strongest non-covalent associations known to occur in nature.

To purify the PF3D7_20109-F21 proteins present in the bacterial cell, you will use a Strep-Tactin-agarose resin. The Strep-tag on the PF3D7_20109-F21 protein will bind to the coated resin, while the other cellular protein will pass through the resin. Remember, the Nico(DE3) cells are not only producing the PF3D7_20109-F21 protein, but also the proteins needed for cellular function and survival. Biotin is a compound that is also able to bind to Strep-Tactin and washing the resin with a low concentration solution promotes competition for binding between the biotin and bound proteins for the Strep-Tactin-coated resin. Proteins that are non-specifically bound will have a lower affinity for the Strep-Tactin than biotin and be washed from the column, whereas the Strep-tagged PF3D7_20109-F21 will remain adhered to the coated agarose resin. To elute the PF3D7_20109-F21 protein from the coated resin, a high concentration of biotin is used to out-compete the Strep-tag for binding.

Schematic of affinity separation process. For purification, agarose beads (yellow) are coated with Strep-Tactin (green). When cell lysate is added to the coated agarose beads, Strep-tagged protein of interest (blue) adheres to the beads and other proteins in the lysate (orange) are washed from the beads.

Protocols

Part 1: Induce expression of PF3D7_20109-F21

For timing reasons, the induction steps were completed prior to class. So you understand how the cell pellets you will use for the protein purification, the steps are shown in the video and protocol below.

To ensure the steps included below are clear, please watch the video tutorial linked here: [Bacterial Induction]. The steps are detailed below so you can follow along!

  1. Inoculated 5 mL of TB media containing 50 μg/mL kanamycin with a colony of Nico(DE3) cells transformed with pET-28b(+)_PF3D7_20109-F21.
  2. Incubated the culture overnight at 37 °C with shaking at 220 rpm.
  3. Dilute the overnight culture 1:100 in 1 L of fresh TB media containing 50 μg/mL kanamycin.
  4. Incubate at 37 °C until the OD600 = 0.6 with shaking at 220 rpm.
  5. To induce PF3D7_20109-F21 protein expression, add IPTG to a final concentration of 1 mM.
  6. Incubate at overnight at room temperature with shaking at 100 rpm.
  7. To harvest the cells, centrifuge the culture at 4000 g for 15 min at 4 °C.
  8. Cell pellets were flash frozen in liquid nitrogen, then stored at -80 °C until used for purification.

In your laboratory notebook, complete the following:

  • Calculate the volume of kanamycin stock that was added to the TB broth in Step #1. In Step #3.
    • Concentration of kanamycin stock = 50 mg/mL.
  • Calculate the volume of IPTG stock that was added to the TB broth in Step #5.
    • Concentration of IPTG stock = 100 mM.

Part 2: Purify PF3D7_20109-F21 protein

To ensure the steps required for purifying the PF3D7_20109-F21 protein are clear, the Instructor will provide a live demonstration of this process.

Lyse Nico(DE3) cells expressing pET-28b(+)_PF3D7_20109-F21

  1. Retrieve the Nico(DE3) pET-28b(+)_PF3D7_20109-F21 cell pellet from the -80 °C freezer and leave it on your bench to thaw.
  1. Add the cell lysis buffer and components to each cell pellet.
    • B-Per bacterial extraction reagent at 4 mL / g of cell pellet
    • lysozyme at 2 μL / mL of B-Per bacterial extraction reagent
    • DNAse I at 2 μL / mL of B-Per bacterial extraction reagent
    • AEBSF to a final concentration of 1 mM
  2. Solubilize the cell pellet in lysis buffer and vortex to mix.
  3. Incubate cell pellet in lysis buffer at room temperature for 15 min.
  4. To pellet the cell debris, centrifuge the lysate at 15,000 g for 30 min at 4 °C.
  5. Complete Part 3: Electrophorese confirmation digests during the centrifugation.

Prepare Strep-Tactin affinity column

  1. Obtain a 500 μL aliquot of 50% slurry (Ni-NTA resin) and mix the slurry by inverting the tube several times.
    • The slurry is the Ni-NTA column matrix!
  2. Centrifuge the slurry for 30 sec then remove the supernatent.
  3. To wash the slurry, add 500 μL of 1X PBS and invert the tube 3 times.
  4. Add the slurry to the column and allow the 1X PBS to run through the column.
    • Be sure a beaker is placed under the column to collect the waste!
  5. When the PBS has flowed through, cap the bottom and the top of the column until you are ready to add the cell lysate.

Purify PF3D7_1351100 from cell lysate

  1. Transfer the supernatent from the centrifuged cell lysate to a fresh microcentrifuge tube.
    • Label the microcentrifuge tube containing the cell pellet as "pellet" and give it to the Instructor! This pellet will be used later when protein expression and purity are examined.
  2. Aliquot 30 μL of the supernatent (from Step #1) to a fresh microcentrifuge tube.
    • Label the microcentrifuge tube containing the aliquot as "lysate" and give it to the Instructor! This aliquot will be used later when protein expression and purity are examined.
  3. Pipet the remaining supernatent into the prepared Ni-NTA affinity column.
    • Be sure that the bottom of the column is capped!
  4. Attach the cap to the top of the column and incubate on the nutator for 2 hrs at 4 °C.
  5. Following the incubation, clamp the affinity column into the ring stand.
  6. Collect the flowthrough from the affinity column.
    • Hold a microcentrifuge tube under the column, then remove the bottom cap from the column and collect the liquid that leaves the column.
    • Label the microcentrifuge tube as "flowthrough" and give it to the Instructor! This aliquot will be used later when protein expression and purity are examined.
  7. To wash the Ni-NTA affinity column, add 10 mL of wash buffer.
    • Hold a microcentrifuge tube under the column, then remove the bottom cap from the column and collect ~250 μL of the liquid that leaves the column.
    • Label the microcentrifuge tube as "wash" and give it to the Instructor! This aliquot will be used later when protein expression and purity are examined.
  8. To elute the PF3D7_1351100 protein from the affinity column, add 1 mL of elution buffer.
    • Hold a microcentrifuge tube under the column, then remove the bottom cap from the column and collect the entire 1 mL of the liquid that leaves the column.
    • Label the microcentrifuge tube as "elution 1" and give it to the Instructor! This aliquot will be used later when protein expression and purity are examined.
  9. Repeat the elution procedure in Step #8 a total of three times.
    • Label the microcentrifuge tubes as "elution 2" and "elution 3" then give to the Instructor! These aliquots will be used later when protein expression and purify are examined.
  10. Lastly, resuspend the slurry from the Ni-NTA affinity column in 250 μL 1X PBS and transfer to a fresh microcentrifuge tube.
    • Label the microcentrifuge tube as "slurry" and give it to the Instructor! This aliquot will be used later when protein expression and purity are examined.

In your laboratory notebook, complete the following:

  • At several steps in the protein purification procedure, samples are collected that will be used later when protein expression and purity are examined. Consider why each of the samples listed below are saved as controls to measure the success of the purification.
    • The pellet from Step #1.
    • The lysate from Step #2.
    • The flowthrough from Step #6.
    • The wash from Step #7.
    • The slurry from Step #10.
  • What is occurring during the incubation in Step #4?

Part 3: Electrophorese confirmation digests

Electrophoresis is a technique that separates large molecules by size using an applied electrical field and a sieving matrix. DNA, RNA and proteins are the molecules most often studied with this technique; agarose and acrylamide gels are the two most common sieves. The molecules to be separated enter the matrix through a well at one end and are pulled through the matrix when a current is applied across it. The larger molecules get entwined in the matrix and are stalled; the smaller molecules wind through the matrix more easily and travel farther away from the well. The distance a DNA fragment travels is inversely proportional to the log of its length. Over time fragments of similar length accumulate into “bands” in the gel. Higher concentrations of agarose can be used to resolve smaller DNA fragments.

Agarose gel loading and electrophoresis. (A) To separate DNA fragments after a digestion reaction, the sample is loaded into the sample slots, or wells, in the agarose. (B) Then an electrophoresis chamber is used to apply an electrical current. The result is that larger sized DNA molecules remain close to the well where the sample was loaded and smaller DNA molecules migrate through the agarose gel. This is due to the negatively charged DNA backbone and position of the electrodes in the electrophoresis chamber.

DNA and RNA are negatively charged molecules due to their phosphate backbone, and they naturally travel toward the positive electrode at the far end of the gel. Today you will separate DNA fragments using an agarose matrix. Agarose is a polymer that comes from seaweed. To prepare these gels, agarose and 1X TAE buffer (Tris base, acetic acid, and EDTA) are microwaved until the agarose is melted and fully dissolved. The molten agar is then poured into a horizontal casting tray, and a comb is added. Once the agar has solidified, the comb is removed, leaving wells into which the DNA samples can be loaded.

For the digests that were prepared in the previous laboratory session, a 1% agarose gel with SYBR Safe DNA stain was used to separate the DNA fragments in the four digest reactions. In addition, a well was loaded with a molecular weight marker (also called a DNA ladder) to determine the size of the fragments.

To ensure the steps included below are clear, please watch the video tutorial linked here: [DNA gel electrophoresis].

  1. Add 5 μL of 6x loading dye to the digests.
    • Loading dye contains bromophenol blue as a tracking dye, which enables you to follow the progress of the electrophoresis.
    • Glycerol is also included to weight the samples such that the liquid sinks into well.
  2. Flick the eppendorf tubes to mix the contents, then quick spin them in the microfuge to bring the contents of the tubes to the bottom.
  3. Load 25 μL of each digest into the gel, as well as 10 μL of 1kb DNA ladder.
    • Be sure to record the order in which you load your samples!
    • To load your samples, draw the volume listed above into the tip of your P200 or P20. Lower the tip below the surface of the buffer and directly over the well. Avoid lowering the tip too far into the well itself so as to not puncture the well. Expel your sample slowly into the well. Do not release the pipet plunger until after you have removed the tip from the gel box (or you'll draw your sample back into the tip!).
  4. Once all the samples have been loaded, attach the gel box to the power supply and electrophorese the gel at 125 V for 45 minutes.
  5. Lastly, visualize the DNA fragments in the agarose gel using the gel documentation system.

Reagents list

  • Terrific broth (TB) (from RPI)
  • kanamycin (from Sigma)
  • isopropyl β-d-1-thiogalactopyranoside (IPTG) (from Sigma)
  • 2x B-Per bacterial protein extraction reagent (from ThermoFisher)
  • lysozyme (from Sigma)
  • DNase I (from Sigma)
  • 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) (from Sigma)
  • phosphate saline buffer (PBS) (from VWR)
  • Ni-NTA agarose (from Qiagen)
  • Wash buffer: 100 mM HEPES (pH = 7.4), 500 mM NaCl, 10 mM imidazole
  • Elution buffer: 100 mM HEPES (pH = 7.4), 500 mM NaCl, 250 mM imidazole
  • imidazole (from Sigma)

Navigation links

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