Team:BrownStanfordPrinctn/Experiments

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JUDGING FORM ⇗

The protocols below were implemented by multiple subprojects. All are documented in Benchling notebooks linked in the Notebooks section.

Designing and assembling constructs: Diagnosis AND Synthesis

Linearizing pET-23b(+)

  1. Make master mix- USE Q5-- 50 μL recipe:
  2. 19 uL qH2O
  3. 2.5 μL forward primer (10μM dilution)
  4. 2.5 μL reverse primer (10μM dilution)
  5. 1 uL Template DNA (10ng plasmid miniprep)
  6. 25 uL of Q5 mastermix

Q5 Thermocycling Procedure:

  1. Initial Denature at 98°C for 30 sec
  2. Denature at 98°C for 10 sec
  3. Annealing at 61°C for 30 sec
  4. used NEB Annealing calculator: https://tmcalculator.neb.com/#!/main, use final concentration of primer in mix (0.5uM)
  5. Extension at 72°C for 35 seconds(Q5 is much faster than Taq, and requires 10 sec per kb)
  6. Go to step 5 X28 times
  7. Final extension at 72°C for 2 min
  8. Hold 10°C forever

Linearizing pBS2E:

  1. Dilute 200uM primer concentration to 10uM
  2. ALIQUOT OF PRIMER: 2ul of primer + 38ul of water ==> 40ul of 10uM
  3. Make master mix- USE Q5-- 50 μL recipe:
  4. 25 ul Q5 master mix
  5. 2.5 μL forward primer (10μM dilution)
  6. 2.5 μL reverse primer (10μM dilution)
  7. 1uL Template DNA (a couple nanograms worth)
  8. 19uL of aliquoted qH2O

Q5 Thermocycling Procedure:

  1. Initial Denature at 98°C for 30 sec
  2. Denature at 98°C for 10 sec
  3. Annealing in a temperature gradient of 72˚C for 30 sec
  4. used NEB Annealing calculator: https://tmcalculator.neb.com/#!/main
  5. use final concentration of primer in mix (not 10uM, actually 0.5uM bc added 1.25 uL of 10uM into a total volume of 25uL)
  6. Extension at 72°C for 155 seconds
  7. Q5 is much faster than Taq, and requires 20-30 sec per kb.
  8. 6.2 kb x 25 seconds = 155 seconds
  9. Cycles: X28 cycles
  10. Final extension at 72°C for 2 min
  11. Hold 10°C forever

PCRing overhangs and his tag onto hG-CSF and teriparatide:

Dilute 200uM primer concentration to 10uM. Using the 10 uM dilution of the primer in the mix.

Make master mix- USE Q5-- 25 μL recipe:

  1. 9.5uL qH2O
  2. 1.25 μL forward primer (10μM dilution)
  3. 1.25 μL reverse primer (10μM dilution)
  4. 0.5uL Template DNA (using originally ordered inserts at 10 ng/uL.)
  5. 12.5 uL Q5 2X Master Mix

Q5 Thermocycling Procedure:

  1. Initial Denature at 98°C for 30 sec
  2. Denature at 98°C for 10 sec
  3. Annealing at 57°C (teriparatide) and 69°C (hG-CSF) for 30 sec
  4. used NEB Annealing calculator: https://tmcalculator.neb.com/#!/main
  5. use final concentration of primer in mix (not 10uM, actually 0.5uM bc added 1.25 uL of 10uM into a total volume of 25uL)
  6. Extension at 72°C for 15 seconds(Q5 is much faster than Taq, and requires 10 sec per kb.)
  7. Go to step 5 x28 times
  8. Final extension at 72°C for 2 min
  9. Hold 10°C forever

Gibson Assembly

  1. Preheat a thermal block to 50C.
  2. Final amount of vector should be 50-100 ng. Divide 100 ng by the vector concentration from Nanodrop to get the volume of vector you should add.
  3. Use https://nebiocalculator.neb.com/#!/ligation to calculate the amount of insert you need. Make sure to put the correct bp lengths for both the vector and the insert. The calculator will give you a result in ng.
  4. Take the nanogram result from the calculator and divide by the insert concentration (from Nanodrop) to get the volume of insert you should add.
  5. If the volume is less than 0.50 uL, make a 1:10 dilution and add 10 times the volume.
  6. Example: we would need to add 0.32 uL of insert. Take 1 uL of the insert and add to 9 uL of H20. In the final tube, you would then add 3.20 uL of the dilution.
  7. Make a table with your samples and the components: Vector, Insert, H20, 2x MM.
  8. The vector volume is calculated from Step 2, and the insert volume is calculated from Step 3. If you are using a dilution, make sure to put the volume of the dilution and not the original volume.
  9. The 2x MM volume is set at 10 uL.
  10. The final volume of all tubes should be 20 uL. Using the other three known volumes, subtract from 20 uL to find the volume of H20 needed.
  11. All components should be on ice, vortexed, and centrifuged. Add them to PCR tubes. Vortex and centrifuge the tubes.
  12. Place tubes on thermal block and incubate for 15 minutes (if doing 2 fragments).
  13. If 4 or more fragments, do 1 hour incubation.
  14. Following incubation, store samples on ice or at -20C for subsequent transformation.

Transformation

  1. Allocate 25ul of NEB 5-alpha or T7 competent cells to centrifuge tubes on ice for 10 minutes.
  2. Add 1ul of chilled assembled product to the centrifuge tubes.
  3. Place the mixture on ice for 30 minutes. Do not mix.
  4. Heat shock for 30 seconds (5-alpha cells)/10 seconds (T7 cells)
  5. Place on ice for 5 minutes. Do not mix.
  6. Pipet 950ul of room temperature SOC into the mixture.
  7. Incubate at 37˚C for 60 minutes and shake at 250rpm.
  8. Warm selection plates with appropriate antibiotics at 37˚C.
  9. Before plating, pipet SOC media and cells up and down to mix evenly.
  10. Plate 50ul, incubate at 37˚C for 12-16 hours.

Protocol for SDS-Page Gels:

Using ThermoFisher Bolt Bis-Tris Plus Mini Gels:

  1. Add loading buffer (2x concentration, in 20ul of sample, add 20ul of loading buffer)
  2. Incubate at 70˚C for 10 min
  3. After 10 min cool samples down on the table and spin to get rid of evaporate from the cup
  4. Rinse gel with DI water after tearing off the foil
  5. Tear off the white strip on the bottom
  6. Green line shows what level to fill up with loading buffer before putting gel in (0.5 L for 1 gel - Bolt MES SDS running buffer: https://www.thermofisher.com/order/catalog/product/B0002)
  7. Pull grey handle to secure the gel make sure the electrodes are positioned on the inside to match lid
  8. Rinse wells with 100ul running buffer
  9. Run at 150 V for 30-40 min
  10. Prepare 100 ml acetic acid 7.5% (100% acetic acid is under the hood) (10 ul aliquots + 50 ml acetic acid (7.5%))
  11. Stop gel. Rinse with DI before opening
  12. Crack the gel open.
  13. Push the gel through the opening where the white tape was
  14. Stain for 30 min (protect from light). Cyber-orange binds to SDS envelope around protein
  15. Shake gently.
  16. Rinse with diH2O
  17. Wash 15min (up to 1h) with acetic acid without stain
  18. Rinse 30 sec with DI H2O just before using the scanner

Scan: Filter 526 (or 520 for stronger signal less acuity) excitation 488 nm

Inducing Protein Synthesis: Diagnosis AND Synthesis

Overnight induction with IPTG

  1. Resuspend a single colony in 5ml LB with antibiotic in a 15mL Falcon tube. Let grow overnight at 37o C in a shaking incubator.
  2. Prepare a 1000ml conical flask with 0.5L of LB + antibiotic.
  3. Inoculate flask with 10ml culture of starter culture.
  4. Incubate at 37o C until OD600 reaches 0.4 - 0.6.
  5. Be sure to take uninoculated LB + antibiotic to use as a starting blank.
  6. Measure first OD just after inoculating.
  7. Measure every 0.5 hour until OD reaches 0.2, then check every 15 mins until OD range is attained.
  8. Add IPTG to a final concentration of 0.4mM.
  9. Induce for 2 hours in a shaking incubator at 37o C.
  10. Spin down until a pellet is observed.
  11. Pour off supernatant, and flash freeze at -80o C overnight.

Lysing Using B-PER Reagent:

  1. Pellet bacterial cells by centrifugation at 5,000 x g for 10 mins
  2. Add 4 ml of Thermo Fisher B-PER Reagent per gram of cell pellet. Pipette the suspension up and down until it is homogenous
  3. Incubate 10 - 15mins.
  4. Spin down until pellet is visible. The pellet will contain cell membrane, cell structure and any inclusion bodies that have formed. The supernatant is cell lysate. Save both fractions until it is confirmed that your protein of interest is in the lysate fraction.
  5. Store at 20C.

DIAGNOSIS: Printing with Experimental Lysate:

Suggested printers:

Our team worked with the Canon Pixma MP950 and the Canon Pixma TR4520 to print our proteins. While the MP950 is in general a higher end Canon printer, for this particular purpose it was not well suited. Error messages that halted function (that are apparently systemic to all units) were inhibitory when we were trying to make the printer “forget” about tampered parts of its hardware. Also the ink cartridges had reader chips that most likely contained information about the color in the cartridge, and whether it had been used before. For a project that is looking to tamper with cartridges, this was not an ideal characteristic.

The TR4520, on the other hand, worked extremely well for our needs. It held only two cartridges, color (containing compartments with blue, red, and yellow) ink and black ink, which made it extremely easy to control exactly what was being printed. For example, we left the color cartridge intact, and in our print surrounded where we wanted our protein to be (signified with black ink on our printing template) with a bold blue background. This eased visualization of where our protein should have been printed, and also provided a helpful negative control when our testing liquid ran past its final destination in our microfluidic device. This printer was also extremely economical and easy to attain.

Prepping Cartridge for Lysate Loading:

We decided to commandeer the black ink cartridge for our experimental lysate. This is how we did it:

  1. Locate the lid, and the seam that connects it to the body of the cartridge.
  2. Using a large screwdriver, press into this seam until the cartridge pops off, or can be torn off. Try to make this as clean a separation as possible, as the lid needs to be reattached for adequate loading into the printer.
  3. Remove the white sponge from the cartridge, and clean with DIwater until runoff is clear when sponge is squeezed.
  4. Rinse inside of cartridge and printhead with DIwater. The printhead is the interface between the sponge and the paper to be printed on. Rinse until runoff from all directions is clear.
  5. Let both components dry/ dry off cartridge with paper towel.

Loading Experimental Lysate:

  1. Pipette ~1ml of experimental lysate onto printhead pad embedded on the inside of the cartridge. This will aid flow of protein through printhead.
  2. Pipette ~2ml of experimental lysate onto depressed square shape on white sponge, making sure it’s well saturated.
  3. Pop sponge back into the cartridge, making sure the depressed square on the sponge itself is aligned with the printhead pad.
  4. Put lid back on cartridge, using scotch tape to affix it to the body.
  5. Load cartridge back into the printer.
  6. Go forth and print.

SYNTHESIS: CELL-FREE SYSTEM

A detailed and formatted guide to cell-free system production can be found linked here

SYNTHESIS: CELLULAR LYOPHILIZATION

  1. Culture cells overnight in its proper broth medium (E. coli, LB; B. subtilis, LB; Vmax, 2xYTP) supplemented with proper antibiotic
  2. Next day, spin down cells in centrifuge. Remove supernatant.
  3. Resuspend in lyophilization media (dilute cells 1:40)
  4. Prepare 96 well-plate with samples by aliquoting 100uL of sample into each well
  5. Set-up lyophilizer, and lyophilize overnight (ideally 12-48 hrs, depending). Remember to freeze cells beforehand.
  6. After lyophilization is complete, rehydrate time point t=0.
  7. Sterilize an 96-well plate and reagent reservoir.
  8. Retrieve proper broth medium and 0.9% saline solution from fridge.
  9. Fill reservoir with saline solution, and pipette 90uL into five rows of the 96-well plate
  10. Fill one row of lyophilized 96 well-plate with 100uL of broth medium
  11. Use a multichannel pipette and remove 10uL of broth medium into pipette into the first row of the 96 well-plate; mix thoroughly.
  12. Take 10uL of solution from the first row of the 96 well-plate and release the solution into the second row of the 96 well plate
  13. Repeat f. for all five rows - serial dilution is performed.
  14. Prepare a large agar plate for bacteria culture.
  15. Using the multichannel, pipette 10uL from the 5th row of the 96-well plate (this is the most diluted sample), and release the solution near the side of the culture plate
  16. Then pipette 10uL from the 4th row of the 96 well-plate, and release the solution on the agar plate slightly above the 5th row. Repeat for all remaining rows.
  17. Take 10uL from the rehydrated sample on the sample 96 well plate and pipette onto the plate as well. Make sure it is above the least diluted sample row.
  18. Let the samples dry on the plate.
  19. Label plate and seal it with parafilm
  20. Let incubate at 37C overnight.
  21. Next day, take photos and count cells.
  22. Calculate cell viability.

PURIFICATION: EXPRESSION CHIP MANUFACTURE

SLA 3D printing (1st iteration)

  1. Upload SolidWorks STL file into SLA formlabs “Form 2 Desktop SLA 3D printer”
  2. Use Preform Slicing software to adjust printing parameters, 25 micron layers, create suitable support structures (verified by Preform), select resin type (Formlabs clear resin).
  3. Begin SLA printing, 10-15 hours
  4. Remove structure from printer
  5. Place in isopropyl bath, 20 minutes
  6. Remove structure from isopropyl bath
  7. Clean excess resin/isopropyl off with alcohol wipes/paper towel
  8. Place in UV hardener, 6-8 hours
  9. Remove supports with pliers
  10. Use sandpaper to smooth outer surfaces
  11. Use needle to carefully outline the inner faces of the two structures with uncured resin
  12. Press two halves of the structure together, using a vice
  13. Use UV lamp to cure the two halves together, 12-20 hours

SLA 3D printing (2nd iteration)

  1. Upload SolidWorks STL file into SLA formlabs “Form 2 Desktop SLA 3D printer”
  2. Use Preform Slicing software to adjust printing parameters, 100 micron layers, create suitable support structures (verified by the Preform), select resin type (dental resin).
  3. Begin SLA printing, 10-15 hours
  4. Remove structure from printer
  5. Place in isopropyl bath, 20 minutes
  6. Remove structure from isopropyl bath
  7. Clean excess resin/isopropyl off with alcohol wipes/paper towel
  8. Flow isopropyl alcohol through chip with a syringe, clearing uncured resin out of the channels
  9. Place in UV hardener, 6-8 hours
  10. Remove supports with pliers
  11. Use sandpaper to smooth outer surfaces

Interfacing

  1. Cut four 1-2 inch lengths of PEEK tubing, 1/8 inch diameter
  2. Use sander to flatten the interfacing side
  3. Coat the end of the tube in epoxy (for each tube)
  4. Hold against the interface of the expression chip until hardened, 5 minutes
  5. Attach latex tubing to the ends of each PEEK tubing interface

PURIFICATION: PDMS DEVICE MANUFACTURE

Photolithography

Wafer Preparation

  1. Dehydration bake wafer on a hot plate at 115°C for at least 3 minutes
  2. Rinse wafer in methanol
  3. Blow dry with nitrogen gas
  4. Bake at 95°C for 10 minutes to dry solvent

SU-8-2005 Spinning

  1. Pour SU-8-2005 on wafer, covering at least ⅔ of surface
  2. Spread by spinning 500 rpm, 10 s, 133 rpm/s acceleration
  3. Cast by spinning 3000 rpm, 40 s, 266 rpm/s acceleration
  4. Soft bake on a hot plate at 65°C for 2 minutes, 95°C for 3 minutes, 65°C for 2 minutes
  5. Expose for 20 seconds at 8.4 mW/cm2 (124 mJ/cm^2) with no mask
  6. Bake on a hot plate at 65°C for 2 minutes, 95°C for 4 minutes, 65°C for 2 minutes

Flow Layer

  1. Pour SU-8-2075 generously, spinning bottle while pouring to avoid bubbles and streaking
  2. Spread by spinning 500 rpm, 10 s, 133 rpm/s acceleration
  3. Cast by spinning 1300 rpm, 33 s, 266 rpm/s acceleration
  4. Let sit on flat surface for at least 20 minutes to allow photoresist relaxation
  5. Soft bake on a hot plate at 65°C for 5 min, 95°C for 30 min, 65°C for 5 min
  6. Expose for 65s at 230 mJ/cm^2, split into two exposures separated by 90s, each for 32.5s, place mask over wafer for the exposure
  7. Hard bake on a hot plate at 65°C for 5 min, 95°C for 12 min, 65°C for 5 min
  8. Submerge in SU-8 developer for 10-13 minutes, until the design becomes visible on the wafer
  9. Hard bake from 65-165°C for 2 hours at 120°C/hour

PFOS Treatment of Wafer

  1. Pour 1-2 drops on PFOS into bottle cap, place cap in bell jar
  2. Place wafers in bell jar next to cap, seal and pull vacuum into bell jar for 1 minute
  3. Switch to no pull to stabilize bell jar, let PFOS deposit for 10 minutes before depressurizing
  4. Place wafers in clean petri dish
  5. Pour 20g of 1:10 thinky mixed PDMS onto each wafer, place in over at 80°C for 1 hour and remove PDMS layer

PDMS Fabrication

  1. Blow dry cup with clean air to remove dust, pour Part A and Part B in a 10:1 ratio of A:B with at least 40g of PDMS for each wafer
  2. Mix for 3 minutes in THINKY mixer at 2000rpm and defoam for 3 minutes
  3. Place wafer in aluminum foil wafer holder which wraps around base of wafer and has walls at least 1” tall
  4. Pour 40-45g PDMS on each wafer, place in vacuum chamber and degas for about 45 minutes
  5. Break vacuum briefly if bubbles in PDMS look like they will rise out of wafer holder, then re vacuum
  6. Bake at 80°C for 60 minutes in convection oven or hot plate
  7. Peel PDMS off of wafer, cut devices out using scalpel and cut inlets and outlets with hole punch, make sure to remove PDMS plugs from punched holes

Plasma treatment

  1. Blow dry glass slide and PDMS layer to remove dust
  2. O2 plasma treat glass and PDMS at 150W for 4.5 minutes in air using a PDC-001 Air Plasma Cleaner
  3. Place device on glass substrate and bake for 24 hours