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Experiments | iGEM Michigan

Experiments

Overview of Project Experimental Protocols

This section displays the experiment protocols used during the cloning, expression, and endocytosis of the protein encapsulin nanocompartment.

Chemical Transformation

Chemical transformation is a common way to incorporate plasmid DNA into chemically competent bacterial cells. MSBT used this protocol to incorporate the encapsulin plasmid into E. Coli cells. Plates with ampicillin were used to select for the cells that had taken up the plasmid, which contains ampicillin-resistance.

Materials

  • Comp cells
  • Plasmid DNA
  • Water bath at 42 C
  • SOC media
  • Agar plate

Procedure

  1. Thaw 50 uL aliquot of comp cells in ice bath
  2. Gently mix thawed cells ONLY BY TAPPING, do not pipette or vortex
  3. Add 2-5 uL of DNA, do not mix
  4. Place on ice for 30 minutes
  5. Heat shock cells for 45 seconds at 42 C timing is exact here Use the water bath to heat shock
  6. Place tubes on ice again for 1-2 minutes
  7. Add growth media (SOC or LB) to bring final volume to 500 uL Should be adding about 445 uL if adding 5 uL of DNA in step 3
  8. Incubate at shaking icubator at 37 C for one hour 160-225 rpm
  9. Spin down cells for 2.5 minutes at 1000 rcf (9000 rpm)
  10. Carefully pour off about 80% of supernatant into waste container, leaving 100 uL
  11. Resuspend cells in remaining supernatant
  12. Spread all cells onto plate and grow at 37 C overnight

Dpn1 Digestion

The digestion was used following PCR to break down any remaining plasmid template DNA prior to ligation. Dpn1 targets methylated DNA, resulting in cleavage of only the template, not the PCR product.

Materials

  • PCR product
  • 10X CutSmart™ Buffer
  • Dpn1

Protocol

  • Add 50 μL (all) of PCR product with 10 μL 10X CutSmart Buffer and 1 μL DnpI
  • Found in main room freezer
  • Incubate at 37 C for 1 hour
  • Heat-inactivate DpnI by incubating at 80°C for 20 minutes
  • After running, place it in the fridge to keep until ready to run a gel

Expression, Isolation, and Purification of Encapsulins

The expression, isolation, and purification of the encapsulins was performed in two stages. First, a test protocol was performed. This was followed by a large batch general expression and his-tag purification using a nickel column. This protocol resulted in pure encapsulin proteins that can be used in endocytosis.

Materials

  • 1M solution IPTG
  • Ampicillin
  • LB Media
  • PBS
  • Tris (0.0024228 g / 1 mL)
  • NaCl (0.008766 g/ 1 mL)
  • SDS
  • Chicken egg white lysozyme
  • Bovine DNAsel

Procedure

  • Inoculate a few mL overnight LB with ampicillin (1:1000, stock solution = 100 mg) and shaking at 37 C
  • Overnight stock: add 5 uL amp to 5 mL of LB + colony
  • Next day: inoculate 1 mL of overnight in 50 mL with Amp, place in shaking incubator at 37 C until OD reaches 0.6-0.8
  • Take away 3 mL of grown bacteria and freeze in 15 mL falcon tube (note OD before --> this is preinduction sample)
  • Add 0.1mM (1:10000 of 1M stock IPTG) to induce protein expression and leave shaking at 18C to express protein overnight
  • Use a 1 M solution of IPTG diluted in autoclaved, distilled water
  • For a 45 mL culture, add 4.5 uL of IPTG
  • Next day: Dilute overnight 1:10 in LB to measure OD and record measurement (example: if you measure 0.43 OD, record 4.3 OD/mL)
  • Dilution: (OD preinduced sample*3mL)/(OD from step 5) = some number S --> Add (S x 1000) uL of induced sample in 3 mL PBS
  • Spin both pre-induction and diluted induced sample from step 6 down in 15 mL falcon tube
  • Discard supernatant and carefully take away any additional supernatant
  • Add 1 mL of 20 mM Tris (pH=7.5) and 150 mM NaCl to supernatant
  • Either: a) Sonicate, or b) Freeze-Thaw Method
  • Take out lysate, put into 1.5 mL eppendorf, and spin 8500x g for 10 minutes
  • Take out all of supernatant, add 1 mL of buffer to the pellet and resuspend pellet
  • Run samples on SDS page gel
  • Inoculate a few mL overnight LB with ampicillin (1:1000, stock solution = 100 mg) and shaking at 37 C
  • Overnight stock: add 5 uL amp to 5 mL of LB + colony
  • For a 1 L total make 10 mL of culture
  • For 500 mL total use 5 mL of culture
  • Next day: inoculate 5 mL of overnight in 500 mL with Amp, place in shaking incubator at 37 C until OD reaches 0.8 OD
  • Add 0.1mM (1:10000 of 1M stock IPTG) to induce protein expression and leave shaking at 18C to express protein overnight
  • Use a 1 M solution of IPTG diluted in autoclaved, distilled water
  • For a 500 mL culture, add 50 uL of IPTG
  • Next day: spin down cells, take them out, and put in 50 mL falcon tube Weigh pellet
  • Add 5 mL/g of cells of 20 mM Tris (pH=7.5) and 150 mM NaCl buffer tube of 10x solution in cubboard in rack
  • Add 0.1 mg of chicken egg white lysozyme and 0.1 mg bovine DNAsel/1mL of buffer
  • Resuspend cells (gently until homogenous, will take a hot sec) and then sonicate (or freeze-thaw)
  • SPRINT back and forth approx. 7 times
  • glass of ethanol in -80
  • 42 C for water bath or hot plate with container of water
  • 3 minutes per station
  • Spin down lysate for 20 minutes at 8500X g
  • Add 2 mL of bed volume of Ni-NTA resin to buffer and let bind on rocker for about 2 hours at 4 C slow on side, cold as possible
  • Put lysate + resin on a column and collect flowthrough
  • Flow 10 Column Volumes (bed volume) of 20 mM Tris (pH=8) over the resin (try to not disturb the resin too much)
  • Collect and wash (first wash)
  • Flow 10 CV of 20 mM Tris (pH=8) and 300 mM NaCl and 40 mM Imidazole (pH=8)
  • Don't disturb resin too much and collect (wash 2)
  • Flow 5 CV of 20 mM Tris, pH8 300 mM NaCl, 300 mM Imidazole, pH8 to elute (collect this!!)

Agarose Gel Electrophoresis

Agarose gel electrophoresis is an important validation step during the DNA assembly process. Running an E-gel on one or more samples of DNA allows any fragments of DNA within each sample to be separated according to molecular weight/DNA length/number of base pairs. MSBT utilized this in many points of experimentation following PCR and Gibson assembly. If the amplicon is of a known size, then an E-gel can be used to confirm the success of the PCR if there is a large presence of the amplicon.

Materials

  • Agarose Gel
  • 50ml of 0.5x TBE buffer
  • 0.5g of agarose. (to make a 1% gel)
  • 5uL of GelRed
  • Digested PCR product
  • DNA ladder
  • HyLadder 10 kb
  • Loading dye
  • Gel mold & comb
  • Excess 1x TBE buffer

Procedure

  • Making the Agarose Gel:
  • To a 250 mL Erlenmeyer flask add 50 mL of 0.5x TBE buffer.
  • Place 0.5 g of Agarose into the flask and microwave on high for 25 s intervals until the agarose has dissolved.
  • When the solution has cooled, but not polymerized, add 5 uL of GelRed to the solution and swirl to mix.
  • Pour the solution into a casting tray and place the desired combs into position. Running the Electrophoresis
  • Allow for approximately 30 mins of polymerization time.
  • Fill the buffer chamber with 1x TBE buffer so that the gel is completely submerged.
  • In the far left well, add 8 uL of DNA ladder
  • From the PCR tube containing the product, remove 6 uL and place in a separate tube
  • To the separate tube, add 1 uL of loading dye and mix with pipette
  • In a well not containing the ladder, pipette the 7 uL of dyed product making sure to not disturb the integrity of the gel
  • Place the casting tray into the gel box in the orientation which allows the negatively charged DNA to run towards the positively charged (red) cathode.
  • Run the gel at a constant voltage set to 120V for approximately 30-45 mins or until the dye front has traveled the desired distance, usually about 75% of the way across the gel, checking every so often to ensure the dye does not run too far off the gel.
  • Visualize the stained gel using a standard transilluminator (302 or 312 nm) and image the gel using an ethidium bromide filter

Inverse Fusion PCR

Due to the small size of the alpha factor in comparison to the large plasmid, infusion pcr was used to add the fragment. This is a 2-step PCR process with 3 primers. The best polymerase based on gel electrophoresis results was a high fidelity platinum taq polymerase, as there is a lower error rate. With such a small fragment addition, having a low error rate was crucial to good results.

Materials

  • FOR THE FIRST PCR REACTION:
  • 38.5 μL nuclease free water (enough to get to 50 μL total volume per PCR tube)
  • 1 μL of 10uM (500nM) primer A
  • 1 μL of 10uM (500nM) primer B
  • 1 μL mNeon plasmid DNA
  • MA76 (make sure it includes histag)
  • 5 μL 10x High fidelity PCR buffer
  • 2 μL MgSO4
  • 1 μL dNTP mix
  • 0.5 μL Platinum Taq DNA polymerase
  • FOR THE SECOND PCR REACTION:
  • 38.5 μL nuclease free water (enough to get to 50 μL total volume per PCR tube)
  • 1ul of 10uM (500nM) primer B
  • 1ul of 10uM 500nM primer C
  • 1 nM (NOT uL!!!) PCR rxn product from the first PCR
  • 10pM original DNA template
  • MA76 (make sure it includes histag)
  • 5 μL 10x High fidelity PCR buffer
  • 2 μL MgSO4
  • 1 μL dNTP mix
  • 0.5 μL Platinum Taq DNA polymerase

Thermocycler Settings

  • Thermocycler Settings Reaction 1 exp 004 on thermocycler
  • Number of cycles: 1
  • Initial denaturation temp: 94 C
  • Initial denaturation time: 30 sec
  • Number of cycles: 30
  • Denaturation temp: 94 C
  • Denaturation time: 15 sec
  • Annealing temp: 55.8 C
  • Annealing time: 30 sec
  • Extension temp: 70 C
  • Extension time: 7.5 minutes
  • Final settings:
  • Final extension: 70 C
  • Final extension time: 10 minutes
  • Hold at 4 C
  • Thermocycler Settings Reaction 2
  • Number of cycles: 1
  • Initial denaturation temp: 94 C
  • Initial denaturation time: 30 sec
  • Number of cycles: 30
  • Denaturation temp: 94 C
  • Denaturation time: 15 sec
  • Annealing temp: 55.8 C
  • Annealing time: 30 sec
  • Extension temp: 70 C
  • Extension time: 7.5 minutes
  • Final settings:
  • Final extension: 70 C
  • Final extension time: 10 minutes
  • Hold at 4 C

Protocol

  • Get ice, all materials should be kept cold
  • Found in storage room, grab a plastic bucket near ice machine to fill
  • Program thermocycler
  • In an eppendorf (1.5 mL, autoclaved) tube, combine all of the elements in the materials section, leaving TAQ polymerase to the end
  • Be sure to place materials back on ice when you are finished
  • Wearing gloves, get a PCR tube and place 50 μL of the master mix into the tube
  • Place in thermocycler and run
  • After running, place it in the fridge to keep until ready to run gel electrophoresis

Chemically Competent Cells

In order to successfully complete chemical transformation, E. Coli cells have to be made chemically competent. The following protocol describes the process to modify the cells.

Materials

  • Overnight culture
  • LB
  • 100 mM CaCl2
  • make sure it is cold
  • Use 0.5549g CaCl2 per 50mL
  • 80% glycerol

Procedure

  • Create liquid growth culture in 500 uL of overnight culture and 50 mL of LB
  • Icubate at 37 C and measure OD until it reaches about 0.5
  • Split growth culture into two 50 mL falcon tubes
  • Centrifuge cells at 3000 x g for 15 minutes at 4 C
  • Make sure centrifuge is balanced and rotor is secure!
  • Pour off supernatent
  • Resuspend cells in 25 mL of 100 mM CaCl2
  • Combine both tubes into one and place on ice for 30 minutes
  • Create a balance tube for the centrifuge
  • Centrifuge cells at 3000 x g for 15 minutes at 4 C
  • Pour off supernatent
  • Resuspend cells in 400 uL of cold 100 mM CaCl2 and 75 uL of 80% glycerol
  • Freeze 50 uL aliquots at -80 C

SDS-PAGE Gel Electrophoresis

SDS-PAGE gels are used for protein presence and size evaluation. This was useful in order to identify the presence of encapsulin protein following expression and purification.

Protocol

  1. Keep samples cold
  2. Prepare a gel tank (use the green ones if you made your own gel; use the black ones if using a pre-made gel) a. Insert gel slide (facing outward if pre-made, facing inward if you made it) b. Fill the tank with running buffer i. 10X running buffer is stored on the shelf above the gel tanks ii. Add 100 mL to a large flask and dilute to 1X with ddH 2 O
  3. Take a 1.5 mL Eppendorf tube for each of your elution samples
  4. Add 8 L of sample to each tube, followed by 2 L of 6X dye (stored at -20 o C)
  5. Heat tubes at 95 o C for 5 min.
  6. Spin in small centrifuge at 12000 rpm for 5 min.
  7. Prepare to load gel plate a. Remove bubbles from each of the wells by taking some of the running buffer and pipetting it into each well until bubbles are gone b. Get protein ladder from -20 freezer
  8. Add 1-2 L protein ladder to the far left well, followed by 10 L flow-through sample (well 2)
  9. Load 10 L of the remaining samples into the other wells and add another 2 L protein ladder at the end
  10. Put lid on top of tank, plug red and black wires into their corresponding outlets
  11. Turn on machine, set time for 1 hour and power (?) constant 25 (?)
  12. At the end of 1 hour, turn off machine and remove plate from the tank
  13. Pry plate open and trim gel to the size that you need
  14. Place gel in the bottom of a recycled pipette tip box and pour a thin layer of Coomassie dye over it
  15. Let it rock at room temp. for 1 hour
  16. After 1 hour, pour off Coomassie dye into the appropriate bottle and add de-staining solution
  17. Look for dark band on gel between 25 and 35 kDa; this is your protein
  18. Let gel rock with de-staining solution for 2 hours
  19. After 2 hours, pour de-staining solution into waste bottle and pour water over gel
  20. Cover container and let rock overnight

Yeast Culture Preparation

Baker's yeast was grown in YPD to be used in future endocytosis experimentation.

Materials

  • 50g YPD broth powder
  • 1L purified H2O

Protocol

  • Make and autoclave new YPD broth with amounts in materials above
  • Incoulate baker's yeast in YPD broth
  • Leave shaking at 32 C and 300 rpm

Endocytosis into Yeast

Yeast was first incubated in YPD growth medium before the introduction of the encapsulin protein. Using a fluorescent microscope for visualization, the encapsulin was added to the yeast. Using mNeon green as a tracker, the location and interactions of the encapsulin protein with the yeast is observed.

Materials

  • Yeast grown in YPD broth
  • Purified encapsulin protein
  • Fluorescent microscope

Protocol

  • On a clean plate, place some of the purified protein on a sample of the yeast culture
  • Incubate for 1 hour at 37 C
  • Transfer a sample to microscope slide and image using fluorescence microscope
  • Look for fluorescence of protein in relation to the yeast cells
  • Protein lacking alpha factor should not endocytose