CRISPRi

Inoculation for culture

1. Add 5ml of Luria Broth (LB) to a culture tube
2. Add 5ul of antibiotic to the culture tube
3. Collect a colony using a pipette tip and insert it into the culture tube
4. Let the tube shake in the incubator and allow growth for 24 hours

1. Resuspend pellet cells in 250ul Resuspension solution. Transfer the cell suspension to microcentrifuge tube.
2. Add 250 uL of Lysis solution, invert until solution is clear.
3. Add 350ul of Neutralization solution, invert tube 4-6 times.
4. Centrifuge microcentrifuge tube for 5 min at 15600 rpm.
5. Pipet supernatant to the GeneJET spin column, avoid disturbing the white precipitate
6. Centrifuge the GeneJET column for 1 min at 15600 rpm
7. Add 50 uL Wash solution and centrifuge for 1 min at 15600 rpm
8. Discard flow through and place column back in collection tube
9. Add 50 uL Wash solution and centrifuge for 1 min at 15600 rpm
10. Discard flow through and placed column back in collection tube
11. Centrifuge 1 min to remove residual Wash solution.
12. Transfer GeneJET spin column to 1.5 mL centrifuge tube and discard collection tube
13. Add 30 uL of Elution buffer to the center of GeneJET spin column membrane to elute plasmid DNA
14. Incubate 2 minutes at room temperature
15. Centrifuge column in centrifuge tube for 2 minutes
16. Discard column and store purified plasmid DNA at -20°C

1. Pre-incubate 348 well plates at 29°C for 30 minutes before pipetting reactions.
2. Retrieve DNA (stored -20°C) and make DNA stocks for sgRNA, dSpyCas9, and P70a-deGFP:
3. sgRNA: make DNA stocks at 120 nM in water.
4. dSpyCas9 plasmid: make DNA stocks at 20 nM in water.
5. P70a-deGFP plasmid: make DNA stocks at 20 nM in water.
6. Retrieve and thaw the sigma 70 myTXTL kits on ice. If the mix is new, vortex gently and spin a few seconds at room temperature in a minifuge, return tube to ice.
7. Keep the tubes on ice, add components in all reactions to myTXTL mix:
8. Add 0.6 μl of dCas9 plasmid stock to the myTXTL mix (1 nM final concentration).
9. For positive control, add 0.6 μl of P70a-deGFP stock to the myTXTL mix (1 nM final concentration).
10. Add 0.5 μl of Chi6 (or GamS) stock to the myTXTL mix (2 μM final concentration).
11. For negative control, add 1.4 μl water to bring the volume to 12 μl.
12. For positive control, add 0.8 μl water to bring the volume to 12 μl.
13. Tap the reaction down a few times.
14. Add .5 μl of a sgRNA stock in each tube (5 nM final concentration). Vortex gently.
15. For each 12 μl reaction, pipette 10 μl into each well of the 348-well plate.
16. Load the pre-warmed plate in the plate reader and start kinetics. On a Synergy HTX Gen 5 plate reader, read the reaction in the settings below.
17. Excitation: 485 nm, Emission at 525 nm,
18. Time lapse between reads: 10 min for 16 h
19. Repeat the entire protocol as necessary for replicates.

Phusion Plus PCR Kit

1. 2X Phusion Plus PCR Master Mix
2. Hydrated template DNA
3. GC enhancer
4. pBest-seq6-s universal primer
5. pBest-seq6-as universal primer
6. Water

Preform PCR: Thermocycler settings

1. Initial Denaturation: 98°C for 30 sec
2. Denaturation: 98°C for 10 sec
3. Annealing: 61°C for 10 sec
4. Extension: 72°C for 30 sec
5. Cycle between denaturation, annealing, and extension for 30 cycles
6. Final extension: 72°C for 5 min
7. Infinite hold

QIAquick PCR Purification Kit

1. Add 5 volumes of Buffer PB to 1 volume of the PCR reaction and mix.
   1. If mixture is orange or violet, add 10 uL of 3 M sodium acetate
2. Place a QIAquick column in a 2 mL tube.
3. Place buffer/DNA mixture into the QIAquick column and centrifuge for 60 sec at max speed. Disregard flow-through.
4. Add 750 uL of Buffer PE to the QIAquick column and centrifuge for 60 sec at max speed. Disregard flow-through.
5. Centrifuge the column once more to remove residual wash buffer.
6. Place the column into a new clean microcentrifuge tube.
7. Add 30 uL of nuclease free water to the column and let stand for 1 minute and then centrifuge.

1. Add 5ml of Luria Broth (LB) to a culture tube
2. Add 5ul of antibiotic to the culture tube
3. Collect a colony using a pipette tip and insert it into the culture tube
4. Let the tube shake in the incubator and allow growth for 24 hours

1. Resuspend pellet cells in 250ul Resuspension solution. Transfer the cell suspension to microcentrifuge tube.
2. Add 250 uL of Lysis solution, invert until solution is clear.
3. Add 350ul of Neutralization solution, invert tube 4-6 times.
4. Centrifuge microcentrifuge tube for 5 min at 15600 rpm.
5. Pipet supernatant to the GeneJET spin column, avoid disturbing the white precipitate
6. Centrifuge the GeneJET column for 1 min at 15600 rpm
7. Add 50 uL Wash solution and centrifuge for 1 min at 15600 rpm
8. Discard flow through and place column back in collection tube
9. Add 50 uL Wash solution and centrifuge for 1 min at 15600 rpm
10. Discard flow through and placed column back in collection tube
11. Centrifuge 1 min to remove residual Wash solution.
12. Transfer GeneJET spin column to 1.5 mL centrifuge tube and discard collection tube
13. Add 30 uL of Elution buffer to the center of GeneJET spin column membrane to elute plasmid DNA
14. Incubate 2 minutes at room temperature
15. Centrifuge column in centrifuge tube for 2 minutes
16. Discard column and store purified plasmid DNA at -20°C

1. Pre-incubate 348 well plates at 29°C for 30 minutes before pipetting reactions.
2. Retrieve DNA (stored -20°C) and make DNA stocks for sgRNA, dSpyCas9, and inhA target construct: a. inhA sgRNA: make DNA stocks approximately 600 nM. b. dSpyCas9 plasmid: make DNA stocks at 20 nM. c. inhA target construct: make DNA stocks at 100 nM.
3. Retrieve and thaw the myTXTL Pro Kit Master Mix on ice. If the mix is new, vortex gently and spin a few seconds at room temperature in a minifuge, return tube to ice.
4. Keep the tubes on ice, add components in all reactions to myTXTL mix: a. Add 0.6 μl of dCas9 plasmid stock to the myTXTL mix (1 nM final concentration). b. For positive control, add 0.6 μl of inhA target construct stock to the myTXTL mix (5 nM final concentration). c. Add 0.5 μl of Chi6 (or GamS) stock to the myTXTL mix (2 μM final concentration). d. For negative control, add 1.4 μl water to bring the volume to 12 μl. e. For positive control, add 0.8 μl water to bring the volume to 12 μl. f. Tap the reaction down a few times.
5. Add .5 μl of a sgRNA stock in each tube (25 nM final concentration). Vortex gently.
6. For each 12 μl reaction, pipette 10 μl into each well of the 348-well plate.
7. Load the pre-warmed plate in the plate reader and start kinetics. On a Synergy HTX Gen 5 plate reader, read the reaction in the settings below. a. Excitation: 485 nm, Emission at 525 nm, b. Time lapse between reads: 10 min for 16 h
8. Repeat the entire protocol as necessary for replicates.

Toehold

1. Pipette toehold stock and nuclease free water into a microcentrifuge tube
2. Briefly vortex and spin dilution down

Phusion Plus PCR Kit

1. Added reagents to a PCR tube according to the reaction chart above.

- One tube for GFP Toehold
- One tube for GFP Trigger

2. Ran tubes in Thermo Cycler for 1 hour using temperatures below:

1. Initial Denaturation: 98°C for 30 sec
2. Denaturation: 98°C for 10 sec
3. Annealing: 61°C for 10 sec
4. Extension: 72°C for 30 sec
5. Cycle between denaturation, annealing, and extension for 30 cycles
6. Final extension: 72°C for 5 min
7. Infinite hold

QIAquick PCR Purification Kit

1. Add 5 volumes of Buffer PB to 1 volume of the PCR reaction and mix.
   1. If mixture is orange or violet, add 10 uL of 3 M sodium acetate
2. Place a QIAquick column in a 2 mL tube.
3. Place buffer/DNA mixture into the QIAquick column and centrifuge for 60 sec at max speed. Disregard flow-through.
4. Add 750 uL of Buffer PE to the QIAquick column and centrifuge for 60 sec at max speed. Disregard flow-through.
5. Centrifuge the column once more to remove residual wash buffer.
6. Place the column into a new clean microcentrifuge tube.
7. Add 30 uL of nuclease free water to the column and let stand for 1 minute and then centrifuge.

1. Preheat the plate reader to 27°C
2. Thaw all kit components and templates at room temperature, then immediately transfer to ice.
3. Directly before use, spin down the myTXTL Pro Master Mix for 1 second with a mini-centrifuge followed by mixing the master mix with a pipette.
4. Add all reagents into a 2mL Eppendorf tube on ice according to the table above.
5. Briefly vortex and mini-centrifuge the assembled myTXTL reaction.
6. Load the pre-warmed plate in the plate reader and start kinetics. On a (Plate reader suggested by paper: Biotek H1m) plate reader, the excitation is fixed at 485 nm, the emission at 525 nm, the time lapse between reads is typically 3 min for 16 h.
7. Repeat the entire protocol as necessary for replicates.

1. Preheat the plate reader to 27°C
2. Thaw all kit components and templates at room temperature, then immediately transfer to ice.
3. Directly before use, spin down the myTXTL Pro Master Mix for 1 second with a mini-centrifuge followed by mixing the master mix with a pipette.
4. Add all reagents into a 2mL Eppendorf tube on ice according to the table above.
5. Briefly vortex and mini-centrifuge the assembled myTXTL reaction.
6. Load the pre-warmed plate in the plate reader and start kinetics. On a (Plate reader suggested by paper: Biotek H1m) plate reader, the excitation is fixed at 485 nm, the emission at 525 nm, the time lapse between reads is typically 3 min for 16 h.
7. Repeat the entire protocol as necessary for replicates.

1. Preheat the plate reader to 27°C
2. Thaw all kit components and templates at room temperature, then immediately transfer to ice.
3. Directly before use, spin down the myTXTL Pro Master Mix for 1 second with a mini-centrifuge followed by mixing the master mix with a pipette.
4. Add all reagents into a 2mL Eppendorf tube on ice according to the table above.
5. Briefly vortex and mini-centrifuge the assembled myTXTL reaction.
6. Load the pre-warmed plate in the plate reader and start kinetics. On a (Plate reader suggested by paper: Biotek H1m) plate reader, the excitation is fixed at 485 nm, the emission at 525 nm, the time lapse between reads is typically 3 min for 16 h.
7. Repeat the entire protocol as necessary for replicates.

Soil Testing

• Soil Sample
• 95% Ethanol
• ZR BashingBead™ Lysis Tube
• ZymoBIOMICS™ Lysis Solution
• Zymo-Spin™ III-F Filter
• ZymoBIOMICS™ DNA Binding Buffer
• Zymo-Spin™ IICR Column
• ZymoBIOMICS™ DNA Wash Buffer 1
• ZymoBIOMICS™ DNA Wash Buffer 2
• ZymoBIOMICS™ DNase/RNase Free Water
• Zymo-Spin™ II-HRC Column
• ZymoBIOMICS™ HRC Prep Solution
• High Speed or Open-Rotor Centrifuge
• Weighboats
• 100-1000ul micropipette and tips
• 1.5 ml Microcentrifuge Tubes

Protocol: Lyse Cells

1. Get a ZR BashingBead™ Lysis Tube for each soil sample and label
2. Collect soil and weigh out 250 mg (.25g)
3. Add 750 μl of ZymoBIOMICS Lysis Solution to each tube
4. Centrifuge the ZR BashingBead™ Lysis Tube for 3 minutes at ≥10,000 g Filter Supernatant
5. Label the Zymo-Spin™III-F Filter and collection tubes for each sample
6. Micropipette 300 ul-400 ul of the supernatant into the ZymoSpin™III-F Filter
7. Centrifuge the collection tube and Zymo-Spin™III-F Filter for 1 minute at 8,000 x g Bind DNA to Column & Wash
8. Discard the Zymo-Spin™ III-F Filter but keep the collection tube (eDNA)
9. Add 800 μl ZymoBIOMICS™ DNA Binding Buffer to the collection tube
10. Add 400 μl 95% ethanol to the collection tube and mix well by pipetting up and down 5-10 times
11. Label a new Zymo-Spin™ IICR Column with a collection tube
12. Add 800 μl of the mixture from step 10 to the Zymo-Spin™ IICR Column (don’t discard remaining liquid)
13. Centrifuge the collection tube and ZymoSpin™ IICR Column for 1 minute at 10,000 x g
14. Discard the liquid that is now in the bottom of the collection tube and place the Zymo-Spin™ IICR Column back inside
    • eDNA is in the column
15. Add the remaining 800 µl into the column and repeat steps 13-14
16. Place the Zymo-Spin™ IICR Column into a new collection tube
17. Add 40 µl ZymoBIOMICS™ DNA Wash Buffer 1 to Zymo-Spin™ IICR Column
18. Centrifuge for 1 minute at 10,000 x g
19. Discard the liquid at the bottom of the collection tube
20. Add 700 µl ZymoBIOMICS™ DNA Wash Buffer 2 to the Zymo-Spin™ IICR column
21. Centrifuge for 1 minute at 10,000 x g
22. Discard the liquid at the bottom of the collection tube, and save the collection tube to put the Zymo Spin™ IICR column back inside.
23. Add 200 µl ZymoBIOMICS™ DNA Wash Buffer 2 to the Zymo-Spin™ IICR column
24. Centrifuge for 1 minute at 10,000 x g
25. Discard the collection tube and keep the Zymo-Spin™ IICR column Elute DNA
26. Transfer Zymo-Spin™ IICR Column to a new 1.5-1.7 ml microcentrifuge tube and label the tube
27. Add 100 µl ZymoBIOMICS™ DNase/RNase Free Water to the Zymo-Spin™ IICR Column and let it sit for 1 minute
28. Centrifuge for 1 minute at 10,000 x g.
    • The water that collects in the 1.5-1.7 ml tube contains the eDNA Filter Final Contaminants
29. Obtain a Zymo-Spin™ III-HRC Filter in a collection tube
30. Add 600 µl ZymoBIOMICS™ HRC Prep Solution to the Zymo-Spin™ III-HRC Filter
31. Centrifuge for r 3 minutes at 8,000 x g
32. Place the Zymo-Spin™ III-HRC Filter in a new 1.5-1.7 ml microcentrifuge tube and label
33. Transfer all of the eDNA in the microcentrifuge tube from step 28 to the Zymo-Spin™ III-HRC Filter
    • It should contain approximately 100 µl
34. Centrifuge for 3 minutes at 16,000 x g
35. Discard the Zymo-Spin™ III-HRC Filter
36. The eDNA is now in the microcentrifuge tube
37. Check quantity of extracted DNA with a NanoDrop spectrophotometer
38. Store eDNA in the freezer

• DNA Concentration from DNA Extraction
• Nuclease Free Water
• PCR Tubes (7 for one sample)
• Microcentrifuge Tube
• EZ PCR Master Mix™, 5X
• 16S Primer Mix
• Primer Mixes of Choice or Integrons
• Control DNA
• Thermal Cycler

1. Dilute eDNA sample 100-fold by adding 2 µl of eDNA Concentration to a microcentrifuge tube with 198 µl Nuclease Free Water
2. Label the tube of diluted DNA with “1/100” and the location of the soil sample
3. Mix well by either flicking the tube or inverting it several times
4. Label 7 PCR tubes A-G
5. Add PCR Reagents to each PCR tube (Refer to table)

Tetracycline PCR

Integron Primer PCR

6. Cap the tubes and flick each tube to ensure the reagents mix well
7. Run tubes in a thermocycler

• 1X TBE Buffer
• Agarose
• DNA stain (e.g., SYBR Safe)
• Fast DNA Ladder 1
• 7 PCR Tubes (A-G)

Protocol: Preparing the Gel

1. Acquire an Erlenmeyer flask, and combine 0.4 g agarose and 20 ml 1X TBE Buffer
2. Swirl into reagents are well mixed
3. Heat solution for about 1 min or until agarose is fully dissolved
4. Add 2 µl of SYBR Safe and swirl until evenly distributed
5. Pour the agarose gel into a casting platform with a gel tray and comb
6. Allow the gel to completely solidify and then remove the comb Running the Gel
7. Place the gel tray with the gel in the buffer chamber
8. Add 30 ml of 1X TBE
9. Load samples onto the gel in the following sequence:
   • Lane 1: 10 µl Fast DNA Ladder 1
   • Lane 2: 15 µl Sample A
   • Lane 3: 15 µl Sample B
   • Lane 4: 15 µl Sample C
   • Lane 5: 15 µl Sample D
   • Lane 6: 15 µl Sample E
   • Lane 7: 15 µl Sample F
   • Lane 8: 15 µl Sample G
10. Cover the gel box and run the gel for about 15-25 mins
11. After the gel, record the results

Cell-Free

1. Make 375 mL 2X YT + P media & Wash Buffer
2. Inoculate BL21 cells into 30 mL of LB and grow for 16 hours in 250mL flask

1. Warm up 2X YT+P 37ºC in incubator for 30 minutes
2. Use 9 mL of 2X YT + P (take from the 375 mL of media) to blank for OD measurement
3. Add 9 mL of cells grown for 16 hours to 375 mL of 2X YT + P media
4. Grow cells to OD600 3.0 ± 2.0 in incubator shaking at 200 rpm at 37ºC

1. Chill big centrifuge to 4 degrees°C
2. Divide 375 mL cell culture grown to OD: add 45 mL of cultured cells to 50 mL falcon tubes until all 375 mL of cell culture are in falcon tubes
3. Centrifuge 1
   1. Centrifuge all falcon tubes for 20 minutes at 3260 XG at 4°C
   2. Keep all tubes on ice after this step
4. While centrifuge is running, weigh and record mass of 1 clean 50 mL falcon tube
5. Dump out supernatant from all falcon tubes
6. Using the spatula, transfer each pellet to the 50 mL Falcon tube, wiping the wet pellet on the side of the Falcon tube to maximize exposed surface area.
7. Resuspend (can vortex) each pellet in 25 mL of wash buffer, vortexing each tube for 20 seconds at a time until suspension is homogeneous (no solid is visible)
8. Centrifuge 4
   1. Balance and centrifuge for 15 minutes at 3260XG at 4°C
9. Store in fridge

1. Dump out supernatant from all falcon tubes

2. Wipe all exposed surfaces of the tube and dry any residual liquid inside the tube

3. Weigh each pellet (if harvested around OD 3.0, each pellet should weigh around 1.5-3 g)

4. Chill a tabletop centrifuge to 4°C

5. Resuspend each pellet in 1 mL wash buffer per gram of cell pellet by vortexing.

6. Let suspensions rest for 5-10 min

7. Aliquot out 1.4 mL of suspension into 2 1.7 mL Eppendorf tube

8. Sonicate suspensions on ice (suspensions should turn brown and become much less viscous)

9. Centrifuge for 15 minutes at 3260 XG and 4°C

10. Pipette off the TOP supernatants (approx. 800 μL from 1.4 mL lysate) & combine into 2 fresh Eppendorf tubes

    1. If lysis has gone well, there will be a separation of the clarified lysate into three bands (clear top band, milky opaque cell debris at the bottom, intermediate band in between)

11. Transfer the supernatant from this spin into a fresh tube (there will be a very small pellet). THIS IS THE FINAL EXTRACT

12. Aliquot into smaller tubes (PCR tubes) at 35 µL each and flash-freeze then store at 80ºC until use

1. Add the following to a 1000 mL flask:

   1. 500 mL distilled water
   2. 4g tryptone
   3. 2.5g yeast extract
   4. 1.25g sodium chloride
   5. 1.75g potassium phosphate dibasic
   6. 0.75g potassium phosphate monobasic

2. Stir well and autoclave.

1. Add the following to a 1000mL flask:
   1. 250 mL tris-base solution
      1. To make a tris-base solutions stock of 1000mL, add 12.11g tris base in 1L of distilled water.
   2. 2.72 g Mg-glutamate
   3. 6.097g K-glutamate
   4. 250 mL distilled water
2. Add 10% acetic acid until pH of the wash buffer is at 7.7. Use a pH probe to measure pH.
3. Stir well, autoclave for 15 minutes at 121ºC.