Experiments

RP4 amplification

For our complete in-vitro replication of GadE, we went through many rounds of primer optimization and testing. We ordered an initial round of primers that were found to have off target effects and poor yield. To improve, we redesigned completely by hand and performed alignments with each primer against RP4 to check for off target annealing. We scanned each primer for disadvantageous secondary structures or dimerization, and once every primer passed these tests it was ordered. Once primers arrived we ran both touchdown PCRs and large scale gradient PCRs for each amplification as well as a variety of different PCR additives such as ETSSB, betaine, and 1,2 propanediol. Through combinatorial testing, we were able to get fragments for all 11 parts of the RP4 assembly with extremely minimal off targets and high yields. We then performed NEB HIFI assembly on the fragments, confirming that size was not an interference with the homology-based assembly.

We have uploaded the primers (BBa_K4598013-BBa_K4598036) that successfully amplify RP4 with minimal off-targets and quality yield. Our iGEM parts database list cycling conditions for each part, as well as additive amounts that worked well.

Conjugation Procedure

Before beginning acid survivability experiments, combining the curli and gadE upregulating plasmids was necessary. However, standard cotransformations utilizing electroporation yielded no success due to the constraints and inefficiencies associated with E. coli Nissle. Alternatively, we chose to proceed with a conjugative approach to combine these vectors. Starting with three separate overnight cultures of EcN + Curli, EcN + gadE, and EcN + RP4 (a conjugative plasmid). Each of these cultures was washed of antibiotics in PBS. After diluting each sample to an OD of 0.5, 1 mL of each was combined and set into a 37*C shaker for 1 hour. As the gadE plasmid contained an oriT site allowing for conjugative transport via RP4, all combinations of the three plasmids should have occurred in the joint culture. We then plated on the antibiotic resistances associated with the gadE and curli plasmids (CAM and KAN respectively) to select for the target combinations.

Positive selection marker-stamping:

Immediately following the Conjugation Procedure listed above with desired plasmids ( One must contain an oriT site and one must be stationary) along with the RP$ conjugative plasmid, plate 1000x or greater dilutions on antibiotic free plates. Once single colonies form, use a felt colonies stamp to lightly stamp antibiotic free plates. This stamp is then used as a template to lightly stamp plates of every possible combination of antibiotic resistances found in desired plasmids. Recording original colony numbers and transferred colony numbers will provide data on conjugative efficiencies of each plasmid in the system.

Diagram of positive selection marker-stamping

Diagram of positive selection marker-stamping

Chemotaxis Assembly:

  1. 0.12 pmol each fragment
  2. 0.06 pmol backbone
  3. 0.75 uL BsaI
  4. 0.25 uL T4 DNA ligase
  5. 3.5 uL T4 ligase buffer
  6. 1 uL ATP
  7. H20 to 25 uL
  8. Cycling: 37C, 5 mins→16C, 5mins: 110 cycles

Chemotaxis Assay:

After completing combinatorial assembly of chemotaxic binders, all combinations in a low concentration sample were transformed via electroporation into neb10b. 50 mL samples of the newly transformed culture were then mira prepped to produce adequate amounts and concentrations of the chemotaxis binder library. This high concentration sample was then transformed into Nissle using the Nissle Electroporation Procedure previously outlined. Outgrowth was immediately plated onto 0.3 % agar gel with kanamycin resistance parallel to a previously established urea gradient starting at 20 mM concentration along with control Nissle. Data will be collected by analyzing growth patterns either selectively towards or away from the gradient.

Large Size Gel electrophoresis

Reagents

  1. 0.3% Agarose
  2. 400mL TAE
  3. 10 000x Sybr gold or other post stain compatible gel
  4. 10ul Loading dye per sample
  5. ~50ng Large DNA
  6. 1:1 v:v H20 to DNA

Pour extremely thick, low percent gel, 0.3% is suitable for most plasmids from 40-60 kb. We recommend aiming for an 80 mL gel if using a standard mini casting tray. Thicker gels can run higher voltages without melting. Since our gel is such a low percentage, it will melt incredibly easily. The gel should also be run in a cold room, which will further reduce melting risk. It is highly unfavorable to run a large gel, as they can develop heating inequality across the gel. The gel should be boiled thoroughly, as underboiling will severely harm visibility and cause smearing in the gel. Visibility is an incredibly difficult variable to maintain here, so every precaution must be taken. Post stain is highly advisable as well, as the presence of intercalators in a precast gel can cause DNA to run differently. Furthermore, post stains like SYBR gold are far more sensitive than ethidium bromide, GelRed, and SYBR safe, allowing less DNA to be run. Lastly, large plasmids can easily cause smearing if too much DNA is loaded, so be conservative. Gel voltage for mini-gel: 35 volts for ~5 hours. Ladder recommended is MidRange PFG marker from NEB or an alternative of similar size.

Chemotaxis Assay:

After completing combinatorial assembly of chemotaxic binders, all combinations in a low concentration sample were transformed via electroporation into neb10b. 50 mL samples of the newly transformed culture were then mira prepped to produce adequate amounts and concentrations of the chemotaxis binder library. This high concentration sample was then transformed into Nissle using the Nissle Electroporation Procedure previously outlined. Outgrowth was immediately plated onto 0.3 % agar gel with kanamycin resistance parallel to a previously established urea gradient starting at 20 mM concentration along with control Nissle. Data will be collected by analyzing growth patterns either selectively towards or away from the gradient.

gadE and csgX

Congo Red Plating Assays Arabinose Induction:

Following the Congo Red and YESCA media Plating Protocol in Protocols, plates were made both with and without arabinose concentrations of 0.1%. These plates were used to run the following three experiments:

The first comprised of two plates (one arabinose induced and one control) grown in 37*C and two plates (one arabinose induced and one control) grown in 30*C. On these plates, E. coli Nissle carrying the curli plasmid was plated in three dilutions of 1x, 10x, and 100x starting at an OD of 0.6. The goal of this experiment was to obtain qualitative data on colony size and color indicative of growth rates and accumulation of Congo Red Dye via binding to curli amyloid fibers. Images and results presented in the results section.

Congo REd Plating Assay Nissle vs DH5α:

Following the Congo Red and YESCA media Plating Protocol in Protocols, plates were made and used to run the following three experiments:

The first comprised of two plates (one arabinose induced and one control) grown in 37*C and two plates (one arabinose induced and one control) grown in 30*C. On these plates, three dilutions of E. coli Nissle without the curli plasmid were added at dilutions of 1x, 10x, and 100x starting at an OD of 0.6. The goal of this experiment was to obtain qualitative data on colony size and color indicative of growth rates and accumulation of Congo Red Dye via binding to curli amyloid fibers. Images and results presented in the results section.

Acid Survivability:

Curli

Three iterations of acid survivability experiments were conducted through plate read growth measurements. These experiments utilized m9 aliquots of pH spanning every half pH value from a pH of 7 to a pH of 1. The first two experiments focused on the curli plasmid expression using pH ranges of pH 7, pH 4, pH 3, pH2, pH1 and pH 4, pH 3.5, pH 3, pH 2.5, pH 2 respectively. These experiments began with cultures of E. coli Nissle carrying the curli plasmid and E. coli Nissle cultures without the plasmid. Both these cultures inoculated two new cultures induced with 0.0015% arabinose and 0% arabinose respectively. All four cultures grew for exactly 12 hours at which point they were each divided into 96-well plates in triplicates for each pH value. All controls were added alongside. Plates were then entered into the plate reader, a set for 24 hour procedures with OD:600 reads spaced every three minutes followed by shaking.

Curli + gadE Conjugation Procedure

Before beginning acid survivability experiments, combining the curli and gadE upregulating plasmids was necessary. However, standard cotransformations utilizing electroporation yielded no success due to the constraints and inefficiencies associated with E. coli Nissle. Alternatively, we chose to proceed with a conjugative approach to combine these vectors. Starting with three separate overnight cultures of EcN + Curli, EcN + gadE, and EcN + RP4 (a conjugative plasmid). Each of these cultures was washed of antibiotics in PBS. After diluting each sample to an OD of 0.5, 1 mL of each was combined and set into a 37*C shaker for 1 hour. As the gadE plasmid contained an oriT site allowing for conjugative transport via RP4, all combinations of the three plasmids should have occurred in the joint culture. We then plated on the antibiotic resistances associated with the gadE and curli plasmids (CAM and KAN respectively) to select for the target combinations.

The third iteration sought to examine the combined effects on acid survivability of upregulated gadE and Curli fiber expression. Following the same acidic media procedure as the previous plate read experiments using pHs of 4, 3.5, 3, 2.5, and 2, we plated the following combinations of plasmids and inducers in this configuration as shown below. Each plasmid-inducer combo along with controls were plated into a 384 well plate and set for a 24 hour procedure with OD:600 reads spaced every three minutes followed by shaking.

Step-by-step Miraprep protocol.

Protocol Title: Miracle-prep (Miraprep)

PROTOCOL FOR: Plasmid DNA isolation

Manuscript Title: The Miraprep: A protocol that uses a Miniprep kit and provides Maxiprep yields

Reagents

Procedure

  1. Set up a 50 ml bacterial culture in appropriate selective media and incubate on a shaker (250 rpm) at 37°C overnight.
  2. Transfer bacteria to a 50 ml tube and spin at 4000xg at 4°C for 10 min.
  3. Discard supernatant and resuspend pellet in 2 ml resuspension buffer with 50 ug/ml RNase freshly added.
  4. Add 2 ml of lysis buffer, invert 3-4 times and incubate for 3 min at RT.
  5. Add 2 ml of neutralization buffer and invert 3-4 times.
  6. Distribute lysate into 1.5 ml Eppendorf tubes (~4 tubes) by pouring, not pipetting.
  7. Spin at 13,200xg at RT for 10 min.
  8. Collect supernatants in a 15 ml tube and discard pellets.
  9. Add 1x volume of 96% ethanol (~5 ml).
  10. Mix thoroughly for 5 sec.
  11. Load the sample-ethanol mix onto 5 spin-columns in three sequential ~700 µl aliquots--after the addition of each aliquot, spin the column 30 sec at 13,200xg.
  12. Discard flow-through.
  13. Repeat step 11 until the entire sample is run through the spin-columns.
  14. Wash with 500µl washing buffer and spin at 13,200xg at RT for 30 sec.
  15. Discard flow-through.
  16. Repeat step 14-15.
  17. Do a final spin at 13,200xg at RT for 1.5 min.
  18. Discard the old tube and put the column onto a new tube.
  19. Add 30-35μl of dH2O and incubate for 2 min at RT.
  20. Spin at 13,200xg for 2 min to elute the DNA from column.
  21. Combine the eluted DNA from all 5 columns in one tube (~175μl).
  22. Measure DNA concentration.
  23. Store samples at -20°C.

Golden Gate Assembly:

Updated: November 18, 2020

Materials

Notes: Super important. Do not freeze & thaw the large tube of T4 Ligase Buffer 10X. This has ATP which is sensitive and all ligations will start to fail. This large tube should be aliquoted into many small PCR tubes with ~8µL of buffer each. When doing a reaction only take one small PCR tube out.

Pmol calculator(REMEMBER IT ASKS A VALUE IN MICROGRAMS, YOU MUST CONVERT FROM NANOGRAMS) https://www.promega.com/resources/tools/biomath/ Calculate picomoles in one uL, then determine how many microliters to add to meet the below ratios

Reagent Concentration Amount (µL)
Vector Backbone 0.06 pmol
insert 0.12 pmol
T4 ligase buffer 10x 2.5
T4 ligase 2000 units/uL 0.25
BsaI 20 units/uL 0.75
H20 Add water to 25uL total -

  1. Set up 25 µl assembly reactions as follows
  2. Mix gently by pipetting up and down 4 times.
  3. Briefly microcentrifuge (1 sec.) to bring material to the bottom of tube.
  4. Transfer to thermocycler and program as follows:
    1. 37°C for 5 min
    2. 16°C for 5 min
    3. go to step 1, 30X
    4. 80°C for 20 min (inactivate both enzymes)
    5. 12°C forever
    6. Clean up reaction with a typical PCR cleanup kit(ELUTE WITH LESS elution buffer than normal, there will not be much DNA in the reaction)
    7. Transform into cells and hope for the best
    8. You may need to drop dialyze

Low-salt LB medium

  1. Deionized H2O, to 950 ml
  2. Tryptone, 10 g
  3. NaCl, 1g
  4. Yeast extract, 5 g
  5. To prepare low-salt LB, combine the above reagents and shake until the solutes have dissolved. Adjust the pH to 8.0 with 5 N NaOH. Adjust the volume of the solution to 1 l with deionized H2O.

NISSLE Transformation

  1. Grow overnight cultures of nissle-1917 in culture media (LB or TB) with respective antibiotic at correct temperature
  2. Dilute cells 50-fold into 25 mL low salt LB media and grow until OD=0.55-0.6
  3. Aliquot into 50 mL falcon tubes or tube of equivalent size
  4. Centrifuge at 4,000 xg at 4C for 10 minutes
  5. Discard supernatant
  6. Resuspend in 5 mL ice cold glycerol

Gel Electrophoresis:

THIS IS FOR 1% GEL, YOU MAY NEED TO ADJUST DEPENDING ON SITUATION

Materials

  1. PCR samples
  2. 240 mg agarose
  3. 30 mL TAEr
  4. 3 μL SYBR Safe
  5. 5 μL dye for each ladder and sample

Procedure

  1. Combine 300 mg agarose and 30 mL TAE
  2. Microwave solution in 20 seconds increments until agarose is fully dissolved, making sure to avoid boiling (a lower intensity setting can help with this)
  3. Add 3 μL SYBR Safe into solution
  4. Insert comb into tray
  5. Cast gel into tray, gel should be less than 1 cm thick
  6. Cover gel with tinfoil, wait 10–15 minutes to harden
  7. Add 5 μL of dye to each 5 μL DNA sample
  8. Prep ladders (1 kb and 100 bp) (4 μL H20, 1 μL ladder, 5 μL dye)
  9. Transfer the full 10 μL volume of each dyed dna sample into a lane (if possible, avoid using the edge lanes and adjacent lanes)
  10. Run gels at 100V
  11. Weigh eppendorf tube and write empty mass on tube
  12. Use razor blade to precisely cut out the band of the sample fragment
  13. Store in marked eppendorf tube, & weigh again. Record full mass on tube as well.
  14. Store in -20℃ overnight

Mini-Prep:

Very nice instructions for this on the instructions card, and everything is labeled. Essentially consists of spinning liquid with a centrifuge, adding something, then removing supernatant or sediment.

Materials

  1. 4 volumes of ≥ 95% ethanol per volume of Monarch Plasmid Wash Buffer 2
  2. Culture to be miniprepped

Procedure

  1. Pellet 1–5 ml bacterial culture (not to exceed 15 OD units) by centrifugation(16000 rcf) for 30 seconds. Discard supernatant, or liquid, and keep the sediment.
  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. 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.
  4. Neutralize the lysate by adding 400 μl of Plasmid Neutralization Buffer (B3) (yellow).(LOCATED IN FRIDGE, MAKE SURE TO KEEP REFRIGERATED) Gently invert tube until color is uniformly yellow and a precipitate forms. Do not vortex! Incubate for 2 minutes.
  5. Clarify the lysate by spinning for 2–5 minutes at 16,000 RCF.
  6. Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through.
  7. 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.
  8. Add 400 μl of Plasmid Wash Buffer 2 and centrifuge for 1 minute.
  9. 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.
  10. Add ≥ 30 μl DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.

Inoculation:

  1. Circle colony and label it with a number/letter
  2. Transfer 5 ml of media into a test tube
  3. If media(LB) does not have an antibiotic, you must add 5 uL of your desired antibiotic(located in freezer in middle slider on the right)
  4. Use a pipette tip to very gently tap on selected colony
  5. Eject pipette tip into test tube with media and antibiotic
  6. Re-parafilm plate and put back in fridge
  7. Place tubes in 37°C shaking incubator for 16 hours, then take out and store in fridge

Antibody Staining of pNeae for Plate Reader

  1. Prepare 4 tubes PER induction amount. I recommend running at least three different induction levels. All steps should be performed in as little light as possible and in a cold room. Perform all centrifugation at 4C. Tubes per induction level: cells only, primary only, secondary only, primary and secondary together
  2. Perform each step for each tube unless otherwise noted
  3. Pellet 10^8 cells after induction(common for yeast is 10^6, but creates extremely weak signal if used for bacteria). Remove supernatant and resuspend in 0.5-1mL PBSA.
  4. Pellet cells again. Remove supernatant and resuspend in 50 μL PBSA
  5. For primary only and primary and secondary together tubes:

Electroporation for Nissle:

  1. Fill bucket with ice
  2. Gather electrocompetent Nissle cells from –80 freezer(one with green handle)
  3. Place Cuvette into the -80*C freezer for ~10 minutes
  4. Gather DNA prepared from 50 mL cultures through the Mira Prep Procedure. Concentrations should be >100 ng/uL
  5. Combine 1-2 µL of your resuspended DNA with 100 µL of electrocompetent Nissle cells and place on ice
  6. LABEL TUBE
  7. Place plates with correct antibiotic in incubator so it's warm when you use it later
  8. Mix gently and transfer to electroporation cuvette (between metal plates), place in cuvette holder and hit “pulse”
  9. Immediately add transformation(WARMED) media(975 uL) (SOB) to the cuvette and transfer recovered cells to a test tube
  10. Leave to shake in the shaking incubator for 30-60 minutes
  11. Remove test tubes from incubator and rest on a tube rack
  12. Label plates to correspond to the tubes
  13. Transfer 100 µL of your transformation to its respective plate(100x and 1000x dilutions are best, so mix 1uL of transformant with 99 uL of sob for 100x, 0.1 uL of transformant with 99.9 uL of SOB for 1000x dilution), pour 10–15 glass beads onto the plate, close the lid, and shake the plate to cover all the afar with the cells (not the lid, just side to side shaking)(MAKE SURE PLATE IS NOT UPSIDE DOWN, SMALLER LID SHOULD BE ON THE BOTTOM)
  14. Pour beads into a used beads bin
  15. Replace lid on plate and place in the incubator upside-down (agar-side up)
  16. Leave plates in the incubator for 12–16 hours
  17. Time is specific, take out and parafilm the plates so they are sealed and place in fridge with the clear doors
  18. Bleach tubes after plating cells

Electroporation (Standard):

  1. Fill bucket with ice
  2. Gather cells from –80 freezer(one with green handle)
  3. Gather DNA from distribution(see other protocol)(NOTE: DISTRIBUTION PLATES DO NOT NEED TO BE KEPT ONE ICE FOR THE COURSE OF THE PROCEDURE. DNA is pretty stable at room temp, but for long term storage it should go back in the -20
  4. Combine 1 µL of your resuspended DNA with 25 µL of your cells and place on ice
  5. LABEL TUBE
  6. Place plates with correct antibiotic in incubator so it's warm when you use it later
  7. Mix gently and transfer to electroporation cuvette (between metal plates), place in cuvette holder and hit “pulse”
  8. Immediately add transformation(WARMED) media(975 uL) (SOB) to the cuvette and transfer recovered cells to a test tube
  9. Leave to shake in the shaking incubator for 30-60 minutes
  10. Remove test tubes from incubator and rest on a tube rack
  11. Label plates to correspond to the tubes
  12. Transfer 100 µL of your transformation to its respective plate(100x and 1000x dilutions are best, so mix 1uL of transformant with 99 uL of sob for 100x, 0.1 uL of transformant with 99.9 uL of SOB for 1000x dilution), pour 10–15 glass beads onto the plate, close the lid, and shake the plate to cover all the afar with the cells (not the lid, just side to side shaking)(MAKE SURE PLATE IS NOT UPSIDE DOWN, SMALLER LID SHOULD BE ON THE BOTTOM)
  13. Pour beads into a used beads bin
  14. Replace lid on plate and place in the incubator upside-down (agar-side up)
  15. Leave plates in the incubator for 12–16 hours
  16. Time is specific, take out and parafilm the plates so they are sealed and place in fridge with the clear doors
  17. Bleach tubes after plating cells

Transformation From Distribution:

  1. Check igem distribution airtable and select target DNA you want more of
  2. With a pipette tip, punch a hole through the foil cover into the 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.
  3. Pipette 10 µL 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.
  4. Transform 1 µL of the resuspended DNA into your desired competent cells(use NEB 10b for training)(see other protocol for transformation), plate your transformation with the appropriate antibiotic(shown in igem distribution airtable), and grow overnight.
  5. Pick a single colony and inoculate broth (again, with the correct antibiotic); grow for 16 hours.
  6. Use the resulting culture to miniprep the DNA AND make your own glycerol stock. We recommend using the miniprepped DNA to run QC tests, such as restriction digests and sequencing.

Plating:

Materials needed:

Procedure: (https://legacy.bd.com/europe/regulatory/Assets/IFU/Difco_BBL/244620.pdf)

  1. Put a large magnetic stir bar into a: clean 2L plastic beaker with handle OR a 1L or 2L glass bottle.
  2. Measure out 25g of LB Broth per Liter (50g for 2L) in a plastic tray on the scale.
  3. Tare the scale again and measure out 15g of Bacto Agar per Liter.
  4. Put the powder into the beaker or bottle and fill it with DI water to 1L or 2L.
  5. If using beaker, place on cold hotplate and using magnetic stirring to dissolve powder. If using bottles, shake very well or use magnetic stirrer.
  6. If using beaker, place aluminum foil over the entire top. If using bottles, place aluminum foil around the top cap such that you can take the top cap off and put in on the bench and the bottom of the cap will not touch the table, only the aluminum foil will.
  7. Put autoclave tape on the aluminum foil.
  8. Label contents and date on autoclave tape.
  9. When putting top on bottle it should be loose, otherwise bottle may explode in the autoclave.(put tape over the lid so it doesn't fall off
  10. Place the beaker or bottle(s) into an autoclave tray.
  11. Autoclave the bottle(s). The duration depends on volume. Typically 1L needs 30min at 121C (not including ramp time). Larger volume needs longer time. You need to account for the fact that large volumes need longer time for entire volume to reach temperature because of the high specific capacity of water. Note that certain autoclave settings may cause the contents to boil over.
  12. If using bottles, while waiting for autoclave prepare a 60C water bath. No need if using beaker(not really necessary)
  13. Take tray out of autoclave (HOT! use gloves)
  14. Make sure autoclave tape has dark stripes on it. If not something went wrong.
  15. (you don't need to use a water bath, can just leave it out on benchtop)If using bottle place it into hot water bath, submerging about 75%. Don't let dirty bath water get near mouth of the bottle. Don't place the bottle into the water bath immediately after taking it out of the autoclave since it will still be very hot and can actually warm up the water bath. Wait for it to cool a bit ~65C. If using beaker place on cold hotplate, very low stirring ~40rpm (make sure stirring is not making bubbles).
  16. Use the IR thermometer to check the temperature of the bottle or beaker.
  17. Wait for the media to cool down to at least 55C to 60C before adding antibiotic. Using 1000X antibiotic add 1mL of antibiotic per Liter of media(if ur making 2L of media, add 2mL). When adding antibiotic make sure you use aseptic technique. So either do this near a flame or in a BSC(the hood in bacterial room).
  18. After adding antibiotic magnetic stir for 1 min (<80rpm so no bubbles) after to make sure antibiotic is mixed in well.
  19. Get your plates and start pouring the media into them, opening one at a time. Pour as little as you can. I usually pour not enough to cover entire petri dish then gently tilt dish so that media covers everything. Avoid bubbles! For regular 100mm plates I usually pour in stacks of 5 plates. If you stack plates it prevents water condensation on the lids.
  20. Wait until plates have solidified and turn them upside down.
  21. Let the plates dry at least 2 hr before using. Fresh plates can be left at RT for a few days or placed into plastic bags (these come with petri dishes) and put into the cold room or fridge.

DPNI digestion of PCR products:

  1. Add 1 uL of DPNI restriction enzyme to finished PCR reactions(50 uL)
  2. Incubate at 37 for 30 minutes
  3. Incubate at 80 for 20 minutes for inactivation

NEB Hifi Assembly

Reagent Length Pmol/uL Total pmol Assembly amounts Positive control amounts
Insert 1 2217 0.071 pmol 0.0 0.7 uL
Insert 2 4351 0.021 pmol 0.0525 2.5 uL
Insert 3 4811 0.015 pmol 0.0495 3.3 uL
Fragment total 0.03-0.3 for <3 fragments, 0.2-0.5 for >3 fragments Less than ~20% of RXN if using straight PCR products
NEBuilder MM 10 uL 10 uL

Curli Procedures:

Transformation of pBbB8k-csg-amylase into E. coli Nissle 1917 through Electroporation:

Preparation of Competent Cells:

  1. A single colony of E. coli Nissle 1917 was picked from a fresh LB plate and inoculated into 5 ml of LB broth, which was incubated overnight at 37°C with shaking at 200 rpm.
  2. The overnight culture was diluted 1:100 into fresh 50 ml LB medium and allowed to grow until it reached an OD600 of 0.5-0.6.
  3. The cells were then chilled on ice for 20 minutes.
  4. Cells were harvested by centrifugation at 4000 x g for 15 minutes at 4°C.
  5. The pellet was washed three times with ice-cold, sterile 10% glycerol.
  6. Finally, the cells were resuspended in 1 ml of 10% glycerol.

Electroporation:

  1. 50 μl of competent Nissle 1917 cells were mixed with 1-2 μl of pBbB8k-csg-amylase plasmid (previously purified).
  2. The mixture was transferred to a pre-chilled electroporation cuvette.
  3. Cells were pulsed using an electroporator set at 2.5 kV, 25 μF capacitance, and 200 Ω resistance.
  4. Immediately after the pulse, 1 ml of LB broth was added to the cuvette.
  5. Cells were recovered by incubating at 37°C with shaking for 1 hour.
  6. Transformants were selected by spreading the recovered cells on LB agar plates containing the appropriate antibiotic (depending on the resistance gene on the pBbB8k-csg-amylase plasmid) and incubating overnight at 37°C.

Induction with Arabinose:

  1. Individual colonies were picked and grown in LB broth with the appropriate antibiotic till mid-log phase.
  2. The culture was then induced with a final concentration of 0.001% arabinose and incubated for an additional 3-5 hours for curli fiber production.

Curli Fiber Staining on Agar Plates using Congo Red Dye:

  1. A 1% solution of Congo Red dye was prepared and filter-sterilized.
  2. This dye solution was added to molten LB agar (cooled to around 50°C) to achieve a final concentration of 0.004%.
  3. The agar was poured onto plates and allowed to solidify.
  4. Transformed Nissle colonies were streaked onto the Congo Red-containing plates and incubated at 30°C for 48 hours.
  5. Colonies producing curli fibers will take up the Congo Red dye, appearing red or pink on the plate.

Curli Fiber Staining in Liquid Culture and Fluorescence Measurement:

  1. Cultures induced with arabinose were grown to the desired time-point, and then 1 ml of the culture was centrifuged to pellet the bacteria.
  2. The pellet was resuspended in 1 ml of a 0.004% Congo Red solution and incubated for 30 minutes at room temperature with gentle shaking.
  3. The cells were washed three times with phosphate-buffered saline (PBS) to remove any unbound dye.
  4. The stained cells were resuspended in 200 μl PBS.
  5. The fluorescence of the stained cells was measured using a plate reader with excitation at 497 nm and emission at 614 nm. The relative fluorescence units (RFUs) were recorded for each sample.

Yesca Congo Red Agar Plates:

YESCA media: YESCA agar plates: YESCA agar is used to induce curli production. Dissolve 1 g/L yeast extract, 10 g/L casamino acids, and 20 g/L agar in water. Pour 25 mL per Petri dish to make normal YESCA plates. For thin YESCA plates used in plug western blots, pour only 15 mL per Petri dish.

0.1% Arabinose: 1mL of Stock (15% dilute Arabinose) in 150 mL of media

Congo Red: 4uL Congo Red Stock solution per 1mL of media

Quantification of CR fluorescence for bacteria prestained on YESCA CR plates.

  1. Spot 4 μL of overnight bacterial culture on the YESCA CR plate. Incubate the plate at 26°C for 48 h.
  2. Recover bacteria from the YESCA CR plate. Wash them in]==[ 50 mM KPi by centrifugation at 12,000 rpm for 1.5 min and resuspend them in 1 mL 50 mM KPi. Adjust the cell density to 1OD600/mL.
  3. Load 100 μL bacteria suspensions onto a 96-well plate reader. Measure the CR fluorescence with a Spectra max M2 plate reader as described above.

Conjugation Procedure

Before beginning acid survivability experiments, combining the curli and gadE upregulating plasmids was necessary. However, standard cotransformations utilizing electroporation yielded no success due to the constraints and inefficiencies associated with E. coli Nissle. Alternatively, we chose to proceed with a conjugative approach to combine these vectors. Starting with three separate overnight cultures of EcN + Curli, EcN + gadE, and EcN + RP4 (a conjugative plasmid). Each of these cultures was washed of antibiotics in PBS. After diluting each sample to an OD of 0.5, 1 mL of each was combined and set into a 37*C shaker for 1 hour. As the gadE plasmid contained an oriT site allowing for conjugative transport via RP4, all combinations of the three plasmids should have occurred in the joint culture. We then plated on the antibiotic resistances associated with the gadE and curli plasmids (CAM and KAN respectively) to select for the target combinations.

Dry Lab Protocols

Binder Creation

Install and use EZBinder, adjusting parameters as specified in the documentation

Recycling

  1. Find the highest pLDDT structure (MONOMER, not complex) and upload it to the remote server
  2. Create a SLURM script based off the standard header (from the EZBinder scripts)
  3. Edit the following line for your particular binder - remember to have a newline between the header and this line, and a newline between this line and the end of the file. Replace 124-124/0 with the actual length of your protein in amino acids (this example is 124 residues). Replace B662-875 with the actual chains you want to bind to (662-875 of chain B in this example)
    1. python /home/username/RFdiffusion/scripts/run_inference.py inference.input_pdb=/home/username/input_pdb/input.pdb 'contigmap.contigs=[124-124/0 B662-875]' diffuser.partial_T=15 inference.num_designs=300 inference.output_prefix=/home/username/output/RFD_out/1/prtldf_batch1_
  4. Switch to the RFDiffusion venv
  5. Submit the RFDiffusion SLURM job
  6. After the RFDiffusion job completes, create a ProteinMPNN script using the RFDiffusion output as the input directory
    1. Edit an EZBinder generated ProteinMPNN script, replacing the pbddir argument with the RFDiffusion output directory specified above
  7. Switch to the ProteinMPNN venv
  8. Submit the ProteinMPNN SLURM job
  9. After the ProteinMPNN job completes, create an ESMFold script using the ProteinMPNN output as the input directory
    1. Edit an EZBinder generated ESMFold script, replacing the pbddir argument with the ProteinMPNN output directory if it is different than the EZBinder script
  10. Upload esm.py
  11. Switch to the ESMFold venv
  12. Submit the ESMFold job
  13. After the ESMFold job completes, download the ESMFold output csv

Partial Diffusion

Follow the instructions for recycling, but use a structure from RCSB as the input instead of the highest pLDDT structure from a previous run

RosettaDock

  1. Prepare your structures for docking by extracting the target into a single pdb and the monomer binder from ESMFold into a single pdb
  2. Concatenate the two files using the prepare_for_dock() function from helpers.py
  3. FastRelax the concatenated structure and upload it to the server
  4. Edit the RosettaDock SLURM scripts
    1. Use the header from a previous job (remember to switch the partition if your previous job used a GPU-intensive program)
    2. Edit the prepack invocation line to use your complex pdb and edit docking:partners if you’re not docking chains A and B
      1. /path/to/username/rosetta_git/Rosetta/main/source/bin/docking_prepack_protocol.default.linuxgccrelease -s /home/username/complex.pdb -docking:partners A_B -docking:sc_min true
      1. Run the RosettaDock prepack script (this should be very fast, and create a file called complex_0001.pdb)
      2. Edit the RosettaDock invocation line to use your packed complex pdb.
        1. /path/to/username/rosetta_git/Rosetta/main/source/bin/docking_protocol.default.linuxgccrelease -s /home/username/complex_0001.pdb -docking:partners A_B -use_input_sc true -nstruct 50000 -out:file:silent dock_rosetta.out
    3. Submit the RosettaDock SLURM job
    4. When the RosettaDock SLURM job completes, download the dock_rosetta.out silent file.
      1. For later analysis, the lines that start with SCORE contain RosettaDock metrics for that structure (there should be 50000 for the above example)