Experiments

Used the Zymo Quick-DNA Tissue/Insect Microprep Kit D6015 to extract DNA from Caddisfly samples. Protocol for DNA extraction (from Zymo): For optimal performance, add beta-mercaptoethanol (user supplied) to the Genomic Lysis Buffer to a final dilution of 0.5%(v/v) i.e., 500 µl per 100 ml.

1. Add specimen(s) to a ZR BashingBead™ Lysis Tube (2.0 mm). Add 750 µl BashingBead™ Buffer to the tube and cap tightly. Note: Generally, no more than 10 mg tissue should be sampled, since larger samples will exceed the DNA binding capacity of the spin column (See Specifications on page 1). Up to 200 µl of whole blood or up to 1.7 x106 cells suspended in 200 µl PBS can also be sampled.
2. Secure in a bead beater fitted with a 2 ml tube holder assembly (e.g., Disruptor Genie™) and process at maximum speed for 10 minutes. Note: Processing time will vary based on sample input and bead beater. Times may be as little as 5 minutes when using high-speed cell disrupters (FastPrep® -24).
3. Centrifuge the ZR BashingBead™ Lysis Tube (2.0 mm) in a microcentrifuge at ≥10,000 x g for 1 minute.
4. Transfer up to 400 µl supernatant to the Zymo-Spin™ III-F Filter in a Collection Tube and centrifuge at 8,000 x g for 1 minute. Discard the Zymo-Spin™ III-F Filter.
5. Add 1,200 µl of Genomic Lysis Buffer to the filtrate in the Collection Tube from Step 4. Mix well.
6. Transfer 800 µl of the mixture from Step 5 to a Zymo-Spin™ IC Column1 in a Collection Tube and centrifuge at 10,000 x g for 1 minute.
7. Discard the flow through from the Collection Tube and repeat Step 6.
8. Add 200 µl DNA Pre-Wash Buffer to the Zymo-Spin™ IC Column in a new Collection Tube and centrifuge at 10,000 x g for 1 minute.
9. Add 500 µl g-DNA Wash Buffer to the Zymo-Spin™ IC Column and centrifuge at 10,000 x g for 1 minute.
10. Transfer the Zymo-Spin™ IC Column to a clean 1.5 ml microcentrifuge tube and add ≥ 20 µl (10 µl minimum) DNA Elution Buffer directly to the column matrix. Centrifuge at 10,000 x g for 30 seconds to elute the DNA.

Use repliQa HiFi ToughMix

PCR Reaction Mix

• 1.25 uL Forward Primer
• 1.25 uL Reverse Primer
• 12.5 uL repliQa HiFi ToughMix (2X)
• 10 uL dH2O
• Single colony (DNA)

PCR Cycle

• 98°C 3 min
• 32 Cycles:
  • Denaturation: 98°C 30s
  • Gradient Annealing for 30s
    • First half of H-Fibroin gene: 55-65.3°C
    • Second half of H-Fibroin gene: 58-65.4°C
  • Extension: 68°C 130s
• 72°C 5 min
• 4°C ∞

Adapted Optimizing Restriction Endonuclease Reactions from NEB

Prepared Restriction Digest Using the Following:

• 5 uL of 200 ng/uL DNA (total 1 ug of DNA)
• 1 uL BamHI
• 1 uL XhoI
• 5 uL rCutsmart Buffer
• 38 uL H2O

Digest for 1 hour at 37°C then deactivate for 15 minutes 65C

Prepared Ligation Using the Following:

• 2 uL T4 ligase buffer
• 10 uL Motif1 diges
• 5 uL pBlueScript
• 1 uL T4 ligase
• 2 uL H2O

Room temp for 1 hour + 10 min 65C inactivation

Using High Efficiency Transformation Protocol using NEB® 10-beta Competent E. coli (High Efficiency) (C3019H/C3019I)

1. Thaw a tube of DH10B Competent E. coli cells on ice for 10 minutes.
2. Add 1 µl of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
3. Place the mixture on ice for 30 minutes. Do not mix.
4. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
5. Place on ice for 5 minutes. Do not mix.
6. Pipette 950 µl of room temperature NEB 10-beta/Stable Outgrowth Medium into the mixture.
7. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
8. Warm selection plates to 37°C. (did not do so had bubbles)
9. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in NEB 10-beta/Stable Outgrowth Medium.
10. Spread 50-100 µl of each dilution onto LB/carb plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.

1. Using a sterile pipette tip select a single colony from your plate.
2. “Dig” the pipette tip into the liquid + selection marker
3. Incubate the culture at 37°C (E. Coli) or 30°C (Yeast) for 12-18 hr in a shaking incubator.
4. After incubation, check for growth, which is characterized by a cloudy haze in the media.
5. For long term storage, you can suspend strain in a glycerol stock.

Adapted from Monarch® Plasmid DNA Miniprep Kit Protocol (NEB #T1010)

1. Pellet 1–5 ml bacterial culture (not to exceed 15 OD units) by centrifugation for 30 seconds. Discard supernatant.
2. Resuspend pellet in 200 μl Plasmid Resuspension Buffer (B1) (pink). Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps.
3. Incubate the culture at 37°C (E. Coli) or 30°C (Yeast) for 12-18 hr in a shaking incubator.
4. Lyse cells by adding 200 μl Plasmid Lysis Buffer (B2) (blue/green). Invert tube immediately and gently 5–6 times until color changes to dark pink and the solution is clear and viscous. Do not vortex! Incubate for one minute.
5. Neutralize the lysate by adding 400 μl of Plasmid Neutralization Buffer (B3) (yellow). Gently invert tube until color is uniformly yellow and a precipitate forms. Do not vortex! Incubate for 2 minutes.
6. Clarify the lysate by spinning for 2–5 minutes at 16,000 x g.
7. Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through.
8. Re-insert column in the collection tube and add 200 μl of Plasmid Wash Buffer 1. Plasmid Wash Buffer 1 removes RNA, protein and endotoxin. (Add a 5 minute incubation step before centrifugation if the DNA will be used in transfection.) Centrifuge for 1 minute. Discarding the flow-through is optional.
9. Add 400 μl of Plasmid Wash Buffer 2 and centrifuge for 1 minute.
10. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If there is any doubt, re-spin the column for 1 minute before inserting it into the clean microfuge tube.
11. Add 12 μl nuclease-free water to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.
12. Nanodrop and record results.

Day -2

1. Thaw strain to be transformed on YPG plate.
2. Prepare DNA for transformation (using appropriate PCR protocol located in dropbox).

Day 1

1. Inoculate each strain to be transformed from the YPG plate into either 5 ml YPD (if step 1) or SC-Ura (if step 2) and grow overnight at 30C.

Day 2

1. For each transformation, start one subculture by diluting 0.2 ml of the overnight culture into either 5 ml of YPD (if step 1) or SC-Ura (if step 2). Grow 4 hrs at 30C (to 5 x 10^6 cells/ml).
2. Turn on 42C water bath and make sure all materials are ready (boiled salmon sperm DNA, DMSO, PEG, water, LiAc at 1M and 0.1M).
3. Harvest cells by centrifuging at 4000 rpm for 5 min. Discard supernatant by pouring, resuspend in 1 ml water and transfer to clean microcentrifuge tube.
4. Centrifuge at 10,000 rpm for 1 min.
5. Aspirate off supernatant and resuspend in 1ml 0.1M LiAc.
6. Centrifuge at 10,000 rpm for 1 min.
7. Aspirate off supernatant and resuspend in 1ml 0.1M LiAc.
8. Centrifuge at 10,000 rpm for 1 min.
9. Aspirate off supernatant and add following solutions in order (directly on top of cell pellet):
   • 0.240 ml 50% PEG
   • 0.035 ml 1 M LiAc
   • 0.025 ml 2 mg/ml ssDNA (boiled 20 min at 99C)
   • 0.036 ml 100% DMSO
   • 0.034 ml DNA
10. Vortex 15 sec to resuspend cells.
11. Incubate for 30 min in 30C incubator.
12. Incubate for 30 min in 42C water bath.
13. Centrifuge at 10,000 rpm for 1 min
14. Discard supernatant and resuspend cells in 0.1 ml water.
15. Plate 0.1 ml on YPD plate. Grow plates at 30C for 14 hours.

Day 3

1. Replica plate from YPD to either SC-URA (if step 1) or 5-FOA (if step 2) plates using velvets.

1. Collect supernatant from step H centrifuging step and add to bead column (noting to add samples evenly along the wall as to keep the bead level for slow and thorough his-tag purification); Collect flow through; Repeat until samples completely passes (By now, all desired proteins should adhere onto beads)
2. Wash beads using salt solution with gradient change. Then wash with iminazole wash solution ( 300µM of Iminazole + sec buffer contents) 3 times; wait 5 mins for every addition and collect flow through (ensure slow flow for thorough wash);
3. For samples that require a protease cut, seal the bottom of the bead column with parafilm and add sec buffer until beads get submerged. Then add on Eppendorf tube worth of enzyme (ULP1 cut enzyme; enzyme also has his-tags); Seal the top with parafilm and then incubate for overnight on rotating plate
4. Condense flow through volume using filtration tubes with right protein kD size such that all proteins ≥certain threshold gets filtered; add flow through and centrifuge until all liquid either gets condensed or flows through; Take bottle out of centrifuge and flip gently periodically to prevent over condensation which could lead to precipitation and loss of protein products; Wash condensing tube using UP water for future reuse.
5. Wash gel filtration column with sec buffer and calibrate/set up the protein purification machine
6. Load samples to column and calculate predicted harvest volume based on kD value of H-L fibroin complex
7. Start harvesting +2ml before predicted harvest volume and note number of AKTA peaks to determine purity of sample empirically; (ideally, there should be a single peak at the harvest volume) if multiple peaks exist near predicted harvest volume, run western blots to determine the correct tube that contains the target protein
8. Measure concentration mg/ml using nanodrop and cover to concentration.