Experiments

First, we put our parts into Level 0 plasmid assembly vectors.

1. Make 2L LB media and 2L LB + agar for plates (plate pouring protocol below)
2. Resuspend the following dry plasmid DNA samples:
• pJUMP29-1A(sfGFP)
• pJUMP29-1B(sfGFP)
• pJUMP29-1C(sfGFP)
• pJUMP29-1D(sfGFP)
• pJUMP49-2A(sfGFP)
• Ribosome Binding Site: BBa_B0030
• Terminator: BBa_B0015
• mCherry
• Promoter: AB_T7
3. Thaw and transform NEB StableCompetent E.coli cells with the resuspended plasmids (see transformation protocol below). Each part should be in its own bacterial culture.
4. After 24 hours of incubation, pick colonies from the selection plate and inoculate LB media to grow liquid culture.(links to protocols coming soon)
5. Miniprep the colonies using a kit of your choice. We used the ProMega PureYield Miniprep kit (see protocol below).
6. Find the DNA concentration of each component using a nanodrop machine.
7. Store the miniprepped plasmids in -20˚C.

For our assembly, we used the Golden Braid protocol.

1. Resuspend powdered IDT products in to concentration specified on the bottle.
• H-fibroin
• L-fibroin SUMO
• PDI-GPx7 Fusion Protein
2. Make 2L LB + Kan + agar solution for plates; Pour plates
3. The four coding sequences are the three IDT products and mCherry. Assemble each one into a Level 1 plasmid vector (protocol below).
• The reaction volume for the assembly will be 20µL.
• Calculate volumes so that the reaction tube contains 135.97ng of coding sequence, and 75ng each of the promoter, RBS, terminator, and respective pJUMP backbone.
• pJUMP29-1A goes with H-fibroin
• pJUMP29-1B goes with L-fibroin SUMO
• pJUMP29-1C goes with the Fusion Protein
• pJUMP29-1D goes with mCherry
• Top up to 20µL by adding an appropriate amount of DI PCR-grade water.
• To each reaction tube, add the following
• 0.5 ul of Hi-T4 DNA ligase
• Add 2 ul of 10x T4 ligase buffer
• 1 ul of BsaI restriction enzyme
• Put the reaction tubes in a thermocycler using appropriate run settings.
• When the run is over, put the assembled Level 1 plasmids into storage at -20˚C.
4. Transform the level 1 plasmids into their own bacterial cultures, using the same protocol. Use about 2-5 µL of DNA for each plasmid sample.
5. Streak on LB + Kan plates; grow overnight and pick colonies for colony PCR to confirm transfection.
6. If level 1 assembly is successful, miniprep the four plasmids and proceed to level 2 assembly
7. Mix all level 1 plasmids with the appropriate backbone for level 2 assembly of the final expression vector.
8. Perform colony PCR to check success of level 2 assembly.
9. Perform plasmid miniprep with streaked cells made during PCR step and store plasmids in -20C˚

Protein Expression in ShuffleT cells

1. Transform SHuffle T7 competent E. coli with the level 2 plasmid prepared earlier, using above transformation protocol.
2. Streak the bacteria on LB + Kan plates to induce expression of plasmid and ensure successful transformation, selecting for transformed cells.
3. After individual colonies appear, perform colony PCR (see above) to ensure plasmid presence
4. If PCR successfully confirms transformation, streak out cells on LB + Kan plates again and add some colonies to five test tubes of 1 L LB + Kan solution for overnight growth
5. Ideally, the SHuffle T7 cells will correctly fold the silk protein with disulfide bonds as they grow and express the genes in the plasmid.

Cell Lysis and Preparing for Protein Purification

1. While cells grow in the liquid culture, prepare for protein purification by making lysis buffer and sec buffer.
• Cell lysis buffer: 25 µM Tris, 500 µM NaCl, 5% glycerol, Beta-me
• Sec buffer for gel chromatography: 500 µM NaCl, 50µM Tris buffer, 5% glycerol (5% in terms of volume), ultrapure water, 2µM DTT
2. Measure cell growth in spectrophotometer. After cell growth reaches the desired OD600, centrifuge in plasmid bottles and discard supernatant
3. Resuspend cell pellets in prepared lysis buffer (15 ml per 3L samples) and pass through hydraulic grinder
• Wash grinder first with anti-bacterial fluids to clean out any potential contamination
• Add some lysis buffer and some sec buffer to completely covert the internal environment of the tubes
• (After wash) flow resuspended cell solution into the grinder and increase pressure slowly until 580 - 800 bar
• Harvest solution after cell solution appear less viscous
4. Collect solution of lysed cells and add to high strength test tubes for high speed centrifuge (13500 g for 1-2 hr)
5. While centrifuging samples, prepare nickel beads by washing beads 3 times in sec buffer

Protein Purification (Must be performed in 4-degrees Celsius)

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.

Materials

• NEB StableCompetent E. coli
• Ice in ice bucket
• Microcentrifuge Tubes
• Heat Block
• Ethanol benchtop

Procedure

1. Warm heat block to 42°C
2. Calculate how many tubes of NEB Stable Competent E. coli cells needed. (minimum 25uL of cells needed per reaction) (they come in 200uL / 50uL tubes)
3. Thaw a tube of NEB Stable Competent E. coli cells on ice for 10 minutes. Once the stock tube is thawed, you cannot re-freeze it or the cells will explode and not be competent. So each tube is 1 time use only
4. Add 1 -2 µl containing 100 pg to 100 ng of plasmid DNA to the cell mixture. (unless you have a ridiculously low DNA concentration, 2 uL will usually be sufficient.) Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex or pipette.
5. Place the mixture on ice for 30 minutes.
6. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix. Place on ice for 5 minutes. Do not mix.
7. Pipette 475 µl of room temperature NEB 10-beta/Stable Outgrowth Medium into the mixture and place at 30°C for 60 minutes. Shake the tube horizontally at 250 rpm or rotate.
8. Warm selection plates to 30°C.
9. Mix the cells thoroughly by flicking the tube and inverting. Then spread 50-100 µl of cells or diluted cells onto a selection plate. Incubate plates 24 hrs at 30°C or overnight at 37°C.

1. 10µl ultra pure water, 2.5µl forward primer, 2.5 µl reverse primer, 10µl enzyme mixture for each sample
2. Pick individual colonies and steak them out first on a separate LB + Kan plate before adding colonies into solution mixture
3. Run in thermocycler using the right settings (I will check Benchling to see what our actual cycle is so I can put it in here)
4. Add loading dye (5µl of 5x purple loading dye)
5. Make 100ml 1x TAE + 1mg agarose mixture, heat and mix until homogenous, and pour into mold with appropriate well divider
6. After gel solidifies, pour 1x TAE buffer to max line and add 5µl of the appropriate ladder on the far left; then add 5µl of each sample to subsequent wells
7. Run at 110V until leading edge almost reaches the end; then image with available machines

Preparing Cells and Lysis

1. Make sure that the Column Wash Solution has been prepared by adding ethanol.
2. Preparing Cells:
1. Centrifuge 1.5ml of bacterial culture for 30 seconds at maximum speed in a microcentrifuge. Discard the supernatant.
2. Add an additional 1.5ml of bacterial culture to the same tube and repeat Step 1.
3. Add 600μl of TE buffer or water to the cell pellet, and resuspend completely.
3. Add 100μl of Cell Lysis Buffer (Blue), and mix by inverting the tube 6 times.
• The solution should change from opaque to clear blue, indicating complete lysis.
• Note: Proceed to Step 3 within 2 minutes. Excessive lysis can result in denatured plasmid DNA.
• If processing a large number of samples, process samples in groups of ten or less.
• Continue with the next set of ten samples after the first set has been neutralized and mixed thoroughly.
4. Add 350μl of cold (4–8°C) Neutralization Solution, and mix thoroughly by inverting.
5. The sample will turn yellow when neutralization is complete, and a yellow precipitate will form. Invert the sample an additional 3 times to ensure complete neutralization
6. Centrifuge at 20000g in a microcentrifuge for 3 minutes.
7. Transfer the supernatant (~800 to 900μl) to a PureYield™ Minicolumn with a pipette. Do not disturb the cell debris pellet.
8. Place the minicolumn into a Collection Tube, and centrifuge at maximum speed (20,000 g) in a microcentrifuge for 30 seconds.
9. Discard the flowthrough, and place the minicolumn into the same Collection Tube.

Washing and Elution

1. Add 200μl of Endotoxin Removal Wash (ERB) to the minicolumn. Centrifuge at 20000g for 30 seconds.
2. Add 400μl of Column Wash Solution (CWC) to the minicolumn. Centrifuge at 20000g for 30 seconds.
3. Transfer the minicolumn to a clean 1.5ml microcentrifuge tube, then add 30μl of elution buffer to the column.
4. Let the column sit for 1 minute.
5. Centrifuge at 20000g for 30 seconds.
6. Store the eluted purified plasmids in -20˚C.

You can also find this protocol on the ProMega website.

iGEM Distribution Kit Parts:

1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that you want. Make sure you have properly oriented the plate. Do not remove the foil cover, as it could lead to cross contamination between the wells
2. Pipette 10µL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. The resuspension will be red, as the dried DNA has cresol red dye. We recommend that you do not use TE to resuspend the dried DNA.
3. Transform 1µL of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate antibiotic* and grow overnight.
4. Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours.
5. Use the resulting culture to miniprep the DNA AND make your own glycerol stock (for further instruction on making a glycerol see this page). We recommend using the miniprepped DNA to run QC tests, such as restriction digests and sequencing.

Twist Biosciences:

• For resuspension, briefly centrifuge the tube or plate before opening and resuspend in nuclease free Tris-EDTA (TE) buffer, pH 8.0 or 10 mM Tris-HCl, pH 8.0 to the desired concentration. A concentration of at least 10 ng/μL is recommended for the stock dilution

IDT Products:

• Use the same protocol as with iGEM Distribution Kit parts, but make sure to use molecular biology / PCR-grade water as millipore DI water can be too acidic.

1. Prepare liquid LB. For example, to make 400 mL of LB, weigh out the following into a 500 mL glass bottle:
• 4g NaCl
• 4 g Tryptone
• 2 g Yeast Extract
• Add dH2O to 400 mL
• Note: If your lab has pre-mixed LB agar powder, use the suggested amount, instead of the other dry ingredients above.
2. Loosely close the cap on the bottle (do NOT close all the way or the bottle may explode!) and then loosely cover the entire top of the bottle with aluminum foil. Autoclave and allow to cool to room temperature.
3. Now screw on the top of the bottle and store the LB at room temperature.
• Note: If you intend to do a mini-prep you will usually want to start 3 mL in a falcon tube, but for larger preps you might want to use as much as a liter of LB in a 2 L Erlenmeyer flask.
4. Using a sterile pipette tip or toothpick, select a single colony from your LB agar plate.
5. Drop the tip or toothpick into the liquid LB + antibiotic and swirl.
6. Loosely cover the culture with sterile aluminum foil or a cap that is not air tight.
7. Incubate bacterial culture at 37°C for 12-18 hr in a shaking incubator.
• Note: Some plasmids or strains require growth at 30°C. If so, you will likely need to grow for a longer time to get the correct density of bacteria since they will grow more slowly at lower temperatures
8. After incubation, check for growth, which is characterized by a cloudy haze in the media.
9. For long term storage, you can suspend bacteria in a glycerol stock.