Universal Protocols

Chemical Transformation

Allowing bacterial cells to uptake desirable DNA using a heat-shock method.

Materials:

• Competent cells

• Plasmid DNA

• Water bath at 42°C

• SOC media

  • Can use LB as backup

• Agar plate

Procedure:

1. Prewarm plates and SOC in an incubator.

2. Thaw one 50 uL aliquot of comp cells in an ice bath.

   1. Comp Cells can be found in the -80 oC freezer. They need to be kept on ice AT ALL TIMES.

3. Begin heating up a water bath to 42°C in preparation for a later step.

4. Gently mix thawed comp cells.

   1. ONLY BY TAPPING, do not pipette or vortex.

5. Add 2-5 uL of plasmid DNA. Do NOT mix.

   1. Quantity will depend on plasmid DNA concentration. If low, add on the higher side. If high, add on the lower side. Should nanodrop the product before this step.

6. Place on ice for 30 minutes.

7. Heat shock cells for 45 seconds at 42°C.

   1. Timing is exact here. Use the water bath to heat shock.

8. Place tubes IMMEDIATELY on ice again for 1-2 minutes.

9. Add growth media (SOC or LB) to bring the final volume to 500 uL.

   1. Should be adding about 445 uL if adding 5 uL of DNA in step 3.

10. Incubate in a shaking incubator at 37 oC for one hour.

    1. 160-225 rpm.

11. Spin down cells for 2.5 minutes at 1000 rcf (9000 rpm).

12. Carefully pour off about 80% of supernatant into a waste container, leaving 100 uL.

    1. Can gently pipet out 400ul of supernatant.

13. Resuspend cells in remaining supernatant.

14. Spread all cells onto a plate and grow at 37 oC overnight.

Dpn1 Digest

When higher amounts of plasmid template must be used in the PCR reaction, it is recommended that the PCR product be digested with Dpn1 (NEB #R0176) in order to destroy the plasmid template before setting up the assembly reaction. Dpn1 cleaves only E. coli Dam methylase-methylated plasmid DNA, but does not cleave the PCR product, since it is not methylated.

Materials: 

• PCR product  

• 10X CutSmart™ Buffer 

• Dpn1

Procedure:

1. For a 10 uL reaction, combine 5-8 uL PCR product, 1 uL 10X CutSmart Buffer and 1 uL (20 units) Dpn1. Mix by pipetting or inverting.

   1. Found in the main room freezer.

2. Incubate at 37°C for 1 hour.

3. Heat-inactivate Dpn1 by incubating at 80°C for 20 minutes.

4. After running, place it in the fridge to keep until ready to run a gel.

Gel Electrophoresis

Agarose gel electrophoresis or E-gels are an important validation step during the DNA assembly process. Running an E-gel on one or more samples of DNA allows any fragments of DNA within each sample to be separated according to molecular weight/DNA length/number of base pairs. This allows the scientist to distinguish and isolate the desired size of DNA after it's been separated from the other fragments in the gel. An instance where this is useful is after a PCR amplification. If the amplicon is of a known size, then an E-gel can be used to confirm the success of the PCR if there is a large presence of the amplicon. 

Materials:

• Agarose Gel

  • 50 ml. of 0.5x TBE buffer

  • 0.5 g of agarose (to make a 1% gel)

  • 5 uL of SYBR Green

• Digested PCR product

• DNA ladder

  • HyLadder 10 kb

• Loading dye

• Gel mold & comb

• Excess 0.5x TBE buffer

Procedure:

1. Add 50 mL of 0.5x TBE buffer to a 250 mL Erlenmeyer flask.

2. Add 0.5 g of Agarose powder into the flask. Microwave flask on high for 30 second intervals until the agarose has dissolved (60-90 seconds total). The powder should no longer be visible in the flask. The flask will be hot, handle carefully. Set up gel mold with appropriate well combs in the meantime.

3. When the solution has cooled to 50 oC (use thermometer or temp. gun), but not solidified, add 5 uL of SYBR stain to the solution. Swirl to mix.

4. Immediately pour the solution from the flask into the gel casting tray.

5. Wait approximately 30 minutes for the gel to solidify. 

6. Once the gel has solidified, carefully remove the comb, loosen the gel tray, and place the gel (in the casting tray) into the gel box in the orientation which allows the (negatively charged) DNA to run towards the positively charged (red) cathode.

7. Pour 0.5x TBE buffer into the gel rig until the gel is completely submerged.

8. Use a p20 or p10 pipettor to load ladder and DNA samples into the wells of the gel. Before starting, gather all PCR products, DNA ladder, and loading dye.

9. In the far left well, add 8 uL of DNA ladder.

10. From the PCR tube containing the product, remove 6 uL and place in a separate tube in order to dye the product.

11. Add loading 1 uL loading dye to the 6 uL product and mix by pipetting up and down.

12. Repeat steps 10 and 11 to dye each PCR product.

13. In the wells to the right of the ladder, pipette the 7 uL of dyed product making sure to not disturb the integrity of the gel. Each PCR product needs to go in its own well. As you load the gel, make note of which sample goes into which well.

14. Once all samples have been loaded, attach a lid to the gel box, and attach the lid to the power supply (NOTE: always make sure that the current is off or paused before inserting or removing a cords from the power supply). Make sure leads are color matched.

15. Set the voltage to 115V and run the gel for about 30-45 minutes. It is advisable to check up on the gel from time to time to make sure that it is proceeding normally and ensure the dye does not run too far off the gel. The desired distance for dye travel is usually about 75% of the way across the gel.

16. When the gel seems to be completed, pause/stop the voltage, disconnect and remove the lid, and take the gel (in its tray) to be imaged. Visualize the stained gel using a standard transilluminator (302 or 312 nm) and image the gel using an ethidium bromide filter.

LB Broth

LB broth is a growth media commonly used for E. coli cells. 

Materials:

• Deionized Water

• 5 g. NaCl

• 5 g. Tryptone

• 2.5 g. Yeast Extract

• 1 L Pyrex Bottle

Procedure:

1. In a 1 L pyrex bottle, add 500 mL of dH2O.

   1. Use graduated cylinder for measuring.

2. Add solid chemicals to the bottle, using filter paper to measure on the scale and swirl after adding each component.

   1. 5 g. of NaCl

   2. 5 g. of Tryptone

   3. 2.5 g. of yeast extract

3. Lightly cover (using cap and/or aluminum foil) and label bottle and autoclave on liquid setting. Make sure the cap is not fully screwed on, i.e. enough that the cap stays on, but it can still move up and down and loosely put autoclave tape over the lid. Make sure the bottle is in a secondary container. Label bottle and set autoclave on liquid setting (called LIQUID 20 on the autoclave). This will take ~50 minutes.

Alternate Procedure:

1. In a 1 L pyrex bottle, add 1 L of dH2O.

2. Add solid chemicals to the bottle, using filter paper to measure on the scale and swirl after adding each component.

   1. 25 g of LB broth.

3. Lightly cover (using cap and/or aluminum foil) and label bottle and autoclave on liquid setting. See step 3 above for more detailed autoclave instructions. 

Overnight Culture

Based on the addgene protocol. Overnight cultures are used to grow a small quantity of bacteria that can be used for growth assays, plate streaking, toxicity assays, etc.

Materials:

• LB broth

• Tube or Flask

• Tin foil

• Plate with desired bacteria

Procedure:

1. Add liquid LB to a tube or flask (~ 5 mL).

2. Add Ampicillin (1uL : 1mL).

3. Take a sterile pipette tip and lightly select a colony (touch the colony with the pipette tip, try not to touch more than one!) from the plate and inoculate the LB.

4. Cover tube/flask with tin foil, making sure it is not airtight. Label overnights clearly!

5. Incubate in a shaking incubator at 37 oC overnight (12-18 hr) at 200 rpm.

6. To store remaining overnight liquid cultures, spin down samples in the falcon tubes at 5000 rpm for 5 mins, decant supernatant in waste bottles, and store falcon tubes containing bacterial pellets at -20°C.

Making Competent Cells

Competent cells are used during transformation to take up new DNA.

Materials:

• Overnight Culture

• LB Broth

• 100 mM CaCl2

  • Cold

  • Use 0.5549 g. CaCl2 per 50 mL.

• 80% glycerol

Procedure:

1. Create liquid growth culture by mixing 500 uL of overnight culture and 50 mL of LB in a 50 mL flask.

2. Incubate in a shaking incubator at 37 C. Measure OD using a spectrophotometer and a clean cuvette about every 20-30 minutes until it reaches about 0.5.

   1. Usually 1.5-2 hours.

   2. Growth is exponential, so be mindful that once you get close, you are very close! Check closer to every 5-10 minutes.

3. Split growth culture into two 50 mL falcon tubes (Should have about 25mL each in them).

4. Centrifuge cells at 3000 x g for 15 minutes at 4 oC.

   1. Make sure the centrifuge is balanced and the rotor is secure.

5. Pour off supernatant.

6. Resuspend cells in 25 mL of 100 mM CaCl2, this should be done quickly with a 1 mL pipette.

   1. Gently pipette up and down

7. Combine both tubes into one new, clean 50 mL falcon tube and place on ice for 30 minutes.

8. Create a balance tube for the centrifuge.

9. Centrifuge cells at 3000 x g for 15 minutes at 4 oC.

10. Pour off supernatant.

11. Resuspend cells in 400 uL of cold 100 mM CaCl2 and 75 uL of 80% glycerol.

12. Freeze 50 uL aliquots at -80 oC.

Pouring Agar Plates

Agar plates are the medium on which bacterial colonies are grown.

Materials: 

• dH2O

• NaCl

• Tryptone

• Yeast extract

• Agar

• Sterile plates

Procedure:

1. Make LB Broth

   1. In a 1 L pyrex bottle, add 500 mL of dH2O and add solid chemicals (5 g. of NaCl, 5 g. of Tryptone, 2.5 g. of yeast extract, 7.5 g. of agar) to bottle, using filter paper to measure on the scale and swirling after adding each component OR in a 1L pyrex bottle, add 1L of dH2O and add solid chemicals to bottle (25 g. Of LB broth base and 37 g. Of agar), using filter paper to measure on the scale and swirling after adding each component.

2. LIGHTLY cover (make sure the cap is not fully screwed on, i.e. enough that the cap stays on, but it can still move up and down) and loosely put autoclave tape over the lid. Make sure the bottle is in a secondary container. Label bottle and set autoclave on liquid setting (called LIQUID 20 on the autoclave). This will take ~50 minutes.

3. After autoclaving, to add antibiotics to your LB. Take aliquots of the antibiotic from the freezer to thaw and add 1 mL per 1 L of agar made (instructions for how to make the antibiotic stock are below, if needed). So 500 mL of LB/agar solution = 500 uL of antibiotic. Add this after.

4. Once the LB container has cooled enough to hold with a heat pad, carefully take empty plates out of the sleeve in a stack on the bench.

5. Lift up the entire stack starting on the bottom plate's lid, and pour LB agar until the bottom surface of the plate is covered.

   1. You cannot set down the stack in your hand, this is to maintain sterility.

   2. Try to pour all plates evenly and avoid bubbles as much as possible.

6. Let plates solidify in stacks of 4-5 and store in the freezer.

7. Also make sure to rinse the LB container as soon as you are done, because any leftover agar will solidify and become very hard to clean out!

Ampicillin Stock Procedure:

1. To make stocks, add 100 mg (0.1 g) of ampicillin to 1 mL of dH2O and vortex well. Make 1 mL aliquots and store in the freezer. Working stock can be stored in a 4°C fridge. Ampicillin stock can be stored in 2-8 oC for up to three weeks and can be stored in -20 oC for 4-6 months.

2. Add 1 mL antibiotic stock per liter of agar (or 100 uL to 100 mL of agar) AFTER autoclaving but before pouring plates Cool down autoclaved LB-agar in a water bath and cool to 45-50 oC before adding ampicillin stock. This is important because ampicillin can be inactivated by heat.

LB Broth Base Procedure:

1. Dissolve 15 g of Bacto agar in 1.0 L of LB medium and sterilize by autoclaving. 

2. Cool to 50°C in a temperature-controlled water bath. 

3. Add 0.50 mL of 100 mg/mL ampicillin. Pour into plates.

4. Note: 3.75 g Agar for 250 LB Broth Base

Protein SDS Gel

SDS Page Gels are used to visualize protein bands. They can be used to help identify if an intended protein is in a certain solution or purification. Proteins will be separated on the basis of molecular weight. 

Materials: 

• 20 mM TRIS

• 100 mM NaCl

• Lysozyme

• Yeast extract

• Agar

• Sterile plates

Procedure:

1. Keep samples cold!

2. Prepare a gel tank (use the green ones if you made your own gel; use the black ones if using a premade gel) 

   1. Insert gel slide (facing outward if pre-made, facing inward if you made it) 

   2. Fill the tank with running buffer

      1. The 10X running buffer is stored on the shelf above the gel tanks. 

      2. Add 100 mL to a large flask and dilute to 1X with ddH 2 O 

3. Take a 1.5 mL Eppendorf tube for each of your elution samples. 

4. Add 8 L of sample to each tube, followed by 2 L of 6X dye (stored at -20 oC).

5. Heat tubes at 95 oC for 5 min. 

6. Spin in a small centrifuge at 12000 rpm for 5 min.

7. Prepare to load gel plate:

   1. Remove bubbles from each of the wells by taking some of the running buffer and pipetting it into each well until bubbles are gone. 

   2. Get a protein ladder from the -20°C  freezer.

8. Add 1-2 L protein ladder to the far left well, followed by 10 L flow-through sample (well 2).

9. Load 10 L of the remaining samples into the other wells and add another 2 L protein ladder at the end.

10. Put the lid on top of the tank, plug red and black wires into their corresponding outlets.

11. Turn on the machine, set time for 1 hour and power constant 25.

12. At the end of 1 hour, turn off the machine and remove the plate from the tank.

13. Pry plate open and trim gel to the size that you need.

14. Place gel in the bottom of a recycled pipette tip box and pour a thin layer of Coomassie dye over it. 

15. Let it rock at room temperature for 1 hour. 

16. After 1 hour, pour off Coomassie dye into the appropriate bottle and add a destaining solution.

17. Look for a dark band on gel between 25 and 35 kDa; this is your protein.

18. Let gel rock with a destaining solution for 2 hours.

19. After 2 hours, pour the destaining solution into a waste bottle and pour water over gels.

20. Cover the container and let it rock overnight.

Protein Purification

This protocol is used to purify proteins from E. coli bacteria. It works on the basis that many E. coli proteins will precipitate out of solution at a temperature of above 85C. In this protocol, dialysis was also used for further purification.

Materials: 

• 20 mM TRIS

• 100 mM NaCl

• Lysozyme

• Ammonium sulfate

• Dialysis buffer

• PCR tubes

Procedure:

1. Resuspend cell pellet of ⅛ of 1L culture in 5 mL of 100 mM NaCl 20 mM TRIS at pH 7.5.

2. Add 0.1 mg/mL lysozyme and incubate on ice for 30 minutes.

3. Aliquot all 5 mLs equally into 3 eppendorf tubes.

4. Freeze thaw 6 cycles between ethanol in -80 and 37 degree water:

5. Put a plastic beaker of 200mLs of ethanol in the -80 oC freezer.

6. Put another small beaker in the 37 oC incubator.

7. Let both come to temperature.

8. Place 3 epi tubes in the freezer. Make sure to not open the door all the way, just open it a crack and slide the tube in and out.

9. Wait until the epi tube liquid is frozen (about 1 minute).

10. Take out and place in a beaker in the 37C incubator. Wait until it thaws completely (2 minutes or so).

11. Repeat 5 more times to lyse cells completely.

5. Spin for 10 min at 14,000 rpm, discard pellet.

   1. To discard pellet, pipette supernatant (liquid) into a fresh tube. Discard the tube with the pellet.

6. Aliquot full volume from eppendorf tubes into 20 pcr tubes and heat to 85 oF for 15 min

   1. Divide volume equally into the 20 pcr tubes

   2. Use the thermocycler at 85 oF for 15 minutes. There should be a setting called "purification" preloaded.

7. Spin 10 min at 14,000 rpm, discard pellets

   1. This will require taking the liquid from the pcr tubes and recombining it into eppendorf tubes so they fit in the centrifuge.

8. Split the supernatant into 10 tubes, each with 200 μL of solution.

9. Add ammonium sulfate in 10% fractions to each of 10 tubes and incubate on ice for 1 hr, flicking every 5 min.

   1. This ended up being 0.014-0.14 g ammonium sulfate increasing in intervals of 0.010g.

   2. Add each mass of ammonium sulfate to each of the 10 tubes. Make sure to label appropriately

10. Spin each of these at 14,000 rpm for 10 minutes, collect pellets, resuspend in a lysis buffer (100 mM NaCl 20 mM TRIS at pH 7.5).

    1. Do not throw out extra solutions. See step 13. 

Dialyzed Solutions:

11. Pipette each of the resuspension solutions into dialysis tubing that has been presoaked in the dialysis buffer. Make sure to clip well.

    1. Dialysis buffer is 2 L of 150 mM NaCl and 20mM TRIS

    2. We are using one long stretch of dialysis tubing because we only have 12 clips. We are clipping in between each 10 samples. Make sure to label the clips with tape and sharpie to keep them apart.

12. Put the filled dialysis tube in the dialysis buffer overnight at 4 oF.

Separate from the Samples that are Being Dialyzed:

13. Concentrate the remaining supernatant from step 10 by running it through the protein concentrator tubes. 5 PCR tubes worth of protein to 100 uL and add 0.9 uL of MES pH 6 buffer, or whatever amount and pH are appropriate to incubate overnight at pH 6. Add TEV to this. Save the concentrator tube to use the next day.

14. Adjust the pH of 5 PCR tubes worth of protein to pH 6 and add TEV.

Project-Specific Protocols

Encapsulin PCR

PCR to isolate encapsulin from vectors for use in Gibson Assembly. These steps need to be performed QUICKLY because as soon as you add the polymerase, the reaction will start.

Materials:

• Reconstitute Dry Primers To 100 uM

• Dilute Primers To 10 uM

• Template DNA

• Q5 Reaction Buffer

• dNTPS

• Thermocycler

Procedure (If Using Pre-Made Master Mix):

1. Get an eppendorf tube that has been autoclaved (in the container covered in foil).

2. Get some ice and keep everything on ice at all times.

3. Set the thermocycler.

   1. See below for details

4. Calculate the concentration of Master Mix needed to reach 50uL final volume.

   1. Will depend on DNA concentration

5. Add forward and reverse primer - 2.5 uL each

6. Add template DNA.

   1. Calculate amount using nanodrop concentration (ng/uL) and total 250 ng

7. Quickly place in the thermocycler and begin.

Procedure (If Making Master-Mix):

1. Get an eppendorf tube that has been autoclaved (in the container covered in foil).

2. Get some ice and keep everything on ice at all times.

3. Set the thermocycler.

   1. See below for details

4. Nuclease-free H2O depends on exact template DNA amount, raising the final volume to 50 uL total.

5. Add 10 uL Q5 reaction buffer

6. Add 1 uL dNTPs.

7. Add forward and reverse primer - 2.5 uL each.

8. Add template DNA.

   1. Calculate amount using nanodrop concentration (ng/uL) and total 250 ng

9. Add 0.5 uL polymerase (only if running all reactions immediately).

10. Quickly place in the thermocycler and begin.

Thermocycler Settings:

1. Initial denaturation.

   1. 98 oC for 30 seconds

2. 25 cycles.

   1. 98 oC for 10 seconds

   2. Tm

   3. 72 oC

Experimental Gibson Assembly

Use Gibson Assembly to insert AMP + Encapsulin sequence for expression and cloning. We will be using an NEBuilder HiFi DNA Assembly Kit.

Materials:

• Cloning Vector

• AMP sequence

• Encapsulin sequence

• NEBuilder HiFi DNA Assembly Master Mix

• Deionized water

Calculations:

• Vector

  • Want 50 ng

  • Concentration = 10.49 ng/uL

  • 50/10.49= 4.8 uL vector

•    AMP

  • Have 13.05 fmol of vector

  • Want 3x amount of AMP (39.15 fmol)

  • molarity = 15.13 nM (nmol/L)

  • 39.15 fmol/15.13 nM = 2.59 uL AMP

•  Encapsulin 

  • Have 13.05 fmol of vector

  • Want 3x amount of enc (39.15 fmol)

  • Concentration = 20.5 ng/uL

  • Convert 39.15 fmol to ng = 23.08 ng

  • 23.08 ng / (20.5 ng/uL) = 1.13 uL enc

Procedure:

1. Put everything on ice to start. Begin by adding the vector to a PCR tube. Then add AMP and encapsulin to the mix. Then add water and master mix.

2. Incubate at 50ºC for 15 minutes.

3. If not transforming right away, store at -20ºC.

Growth Assay

To test how the addition of various plasmids impacts the growth of cells following induction of the lac promoter with IPTG.

Overnights:

1. Prepare eight 5 mL LB overnights, all with identical LB

   1. Uninduced: PET, PET+enc, PET+AMP, PET+enc+AMP

   2. Induced: PET, PET+enc, PET+AMP, PET+enc+AMP

2. Add 50 uL 100 mg/mL ampicillin salt to all of them EXCEPT the PET only

3. Shake overnight at 37 C at 225 rpm in shaking incubator

   1. You need to press "start/stop" to start the heating, you will hear a fan if it is on. Do NOT walk away until temperature has reached 37 C

Induction:

4. Innoculate 100 mL of LB+ampicillin with a 1% inoculum (1 mL sample) of the overnight growth. Incubate at 37 °C (need to press start to heat incubator if it is not already running, you will hear a fan and see the temperature rising on the indicator) while shaking at 220 rpm

   1. In other words, add 1 mL of overnight to 100 mL flask of LB+ampicillin and incubate at 37 °C (need to press start to heat incubator) while shaking at 220 rpm

5. Blank spectrophotometer with 1 mL LB.

6. After 1 hour of growth in the incubator at 37 °C at 225 rpm, take OD in spectrophotometer every 30 min, until you reach an OD between 0.45-0.55

   1. Use a new pipette for each sample you take.

   2. Use a new cuvette for each of the 8 flasks, but after each sample the contents of the cuvettes can be emptied into the liquid waste container and washed out with distilled water.

7. When samples reach an OD of 0.5, add 20 uL IPTG to the 4 induced samples, do NOT add any to the non-induced samples.

Making Growth Curves:

8. Shake at 225 rpm at 37 °C.

9. Take a 1 mL sample and measure the OD600 every 30 minutes of each sample

   1. Ensure when taking a sample to swirl beforehand, the cells can settle and alter readings.

10. Continue to shake at 225 rpm at 37 C between each sample.

11. Aim to continue reading for at least 8 hr.

    1. If the sample has an OD of  >1, dilute the volume in the cuvette with LB to ensure accurate readings.

    2. Record factor of dilution and be consistent across samples.

    3. For example, dilute a sample by placing 0.25 mL and 0.75 mL LB in a cuvette and then multiplying the measurement from the spectrophotometer by 4.

Growth Assay Using CFU

Colony Forming Unit Assays are used along with a growth assay to analyze how a toxic protein expressed in bacteria affects bacterial growth. By counting the number of colonies grown on a plate from a sample of bacteria, the total number of live colonies in a solution can be determined. 

Methods:

1. Create 5 mL overnight cultures in LB containing the appropriate selective agent. 

   1. Incubate the liquid cultures at 37°C with shaking for 16 hours.

2. The following day, create small scale growth cultures by adding a 1% (v/v) inoculum of the overnight cultures to 100 mL of LB containing the appropriate selective agent.

3. When the liquid cultures reach OD600 = 0.4-0.5 , induce the expression of all 

plasmids by adding  20 uL IPTG.

OD600 Growth Curve

1. Immediately upon the addition of IPTG, remove 1 mL aliquots of the growth cultures and check  their OD600. These will serve as T0 data points for the growth curves.

2. Continue removing 1mL samples from the growth cultures every half hour (30 min) and checking their OD600. For every other sample (on the hour) after the T0 sample, remove 100 uL of the aliquot (in the cuvette) and dilute it 1/10  in fresh LB. Multiply the resulting absorbance by the dilution factor to gain a more accurate measurement.

3. Aim to continue reading for at least 8 hr.

   1. If the sample has an OD of >1, dilute the volume in the cuvette with LB to ensure accurate readings. 

   2. Record factor of dilution and be consistent across samples. For example, dilute a sample by placing 0.25 mL and 0.75 mL LB in a cuvette and then multiplying the measurement from the spectrophotometer by 4.

*You will need to either take an additional sample for the CFU assay, or account for this solution when diluting that.

CFU Growth Curve: Prepare a sterile 96 well plate for each culture in the following manner:

1. Place 20 uL of each time point aliquot into the first well of the plate and perform a serial dilution, removing 20 uL each time, and finishing on a 1/107 dilution.

2. Plate 10 uL of each dilution onto a selective plate, resulting in 6 spots. Perform this action 5 times  to create an n=5 technical replicate for each time point.

3. After an overnight growth, count the CFU's at the lowest dilution possible and multiply it by the dilution factor to gain a CFU/mL measurement for each time point.

Positive Control Gibson Assembly Protocol

Use Gibson Assembly to insert AMP + Encapsulin sequence for expression and cloning. We will be using an NEBuilder HiFi DNA Assembly Kit.

Materials:

• Cloning vector

• AMP sequence

• Encapsulin sequence

• NEBuilder HiFi DNA Assembly Master Mix

• Deionized water

Calculations:

Amount of AMP = (100 ng x 1000)/(214 bp x 650 daltons) = 0.7189 pmol

Procedure:

1. Put everything on ice to start. Begin by adding the vector to a PCR tube. Then add AMP to the mix. Then add water and master mix.

2. Incubate at 50ºC for 15 minutes.

3. If not transforming right away, store at -20ºC

Purification Protocol for Toxicity Assays

Toxicity assays are used to assess the effects of a toxic protein on the bacteria they are toxic to. They can help to elucidate the strength of the toxic peptide and determine if the potency of the peptide is retained following purification or other processing methods. 

Procedure: 

1. Resuspend a cell pellet of encapsulins empty and encapsulins with an AMP in 150 mM NaCl and 20 mM pH 7.5 TRIS. Use about 5 mL per ⅛ L culture pellet.

2. Incubate the suspension on ice for 30 min with 0.1 mg/mL lysozyme. It should say something like chicken egg lysozyme or smthn in the -20C freezer and be at 10 mg/mL stock.

3. At this point you should have 2 50 mL falcon tubes, each with a suspension of cells and lysozyme, and one of enc and one of enc + amp. Do 6 freeze thaw cycles between a cup of ethanol in the -80 and 37 degree water in that one incubator right there. You might want to think about aliquoting the liquid into smaller tubes so they freeze and thaw faster for greater efficiency and speed.

How to Freeze/Thaw:

4. Put a plastic beaker of 200mLs of ethanol in the -80C

5. Put another small beaker of water in the 37°C incubator

6. Let both the freezer and incubator come to temperature

7. Place tubes in the freezer. Make sure to not open the door all the way, just open it a crack and slide the tubes in and out

8. Wait until the tubes’ liquid is frozen (about 1 minute)

9. Take out and place tubes in the beaker in the 37C incubator. Wait until it thaws completely (2 minutes or so)

10. Repeat 5 more times to lyse cells completely

11. Spin this homogenate at 14,000 rpm in 1.5 mL eppendorf tubes in the centrifuge in the lab for 10 min

12. Collect supernatants without disturbing pellets. Discard pellets. Collect 8 uL samples for gel later.

13. Heat water to 85 degrees in a large (600mL H20) beaker. Incubate clear lysate in this bath for 15 min.

14. Spin this solution, which should have gone from clear to cloudy, at 14,000 rpm in 1.5 mL eppendorf tubes in the centrifuge in the lab for 10 min. It will be clear again now. Collect 8 uL samples for gel later.

15. Collect supernatants without disturbing pellets. Discard pellets.

16. Collect each of the two proteins in its own 50 mL falcon tube so that all of the protein of each kind is in a single tube. Add glycerol to a final concentration of 10% for each and mix thoroughly but gently. DO NOT VORTEX PROTEIN.

17. Freeze the protein in 1.5 mL aliquots in -80, labeled appropriately.

18. If you have time, run a protein gel of the final protein samples, and the intermediate samples you collected earlier.

Gel Instructions:

19. Wear gloves at all times

20. Get a protein gel out of the box in the fridge. Use the ones from last year, not the super old ones we just got.

21. Remove from packaging, remove plastic white tape strip from back.

22. Make 1X solution of SDS Page running buffer from deionized water from sink and 10X SDS PAGE running buffer from the box dispenser.

23. Put gel in the gel rig, click it in with the frame. Fill to fill line with running buffer.

24. Gently remove comb from the top of the gel

25. Mix 8 uL of each protein sample with 2 uL of the loading dye from the freezer. It is in a screw top tube and says something like 4X protein loading dye on the side in marker. Should smell like sulfur and be yellowish. Do this in PCR tubes.

26. Heat protein and load dye samples to 95 degrees for 5 min in the PCR machine. The samples should be blue when you take them out.

27. Spin all the samples for a min in the PCR tube mini centrifuge.

28. Load 10 uL of Invitrogen prestained protein ladder in the first well of the gel. This is just like loading a DNA gel except vertical

29. Load 10 uL of each protein sample in the subsequent lanes. Label samples.

30. Attach lid and supply 35 mA constant current from the power supply until the ladder and loading dye are ¾ - ⅞ the way down the gel.

31. Remove the gel from the rig, and use a knife or screwdriver to pop each of the little plastic rivets holding the front plastic piece of the gel to the back one, starting in a corner and doing 1 at a time.

32. Trim the bottom of the gel off, below where the protein is. There is a little groove in the back plastic piece that the gel gets stuck in, and removing this part of the gel will prevent it from tearing when you lift it off.

33. Gently crop gel to appropriate size so we can see the protein good after it is stained.

34. Place in the lid of a tips box and cover with coomassie blue. Put in the incubator at 50 rpm and room temperature for 30-45 min, or until it is hard to distinguish the ladder from the blue background.

35. Pour coomassie back into the tube it came from.

36. Cover gel in destain and return to the incubator at the same settings for an hour and a half.

37. Pour destain into the big hazardous waste jug, and cover gel in 40% destain solution and 60% water. Cover the gel so the liquid cannot evaporate away, and return to the incubator at the same settings overnight.

38. Image gel the next day

Restriction Enzyme Double Digest (Nde1, Pac1)

Protocol for cutting plasmid at two sites simultaneously with restriction enzymes NdeI and PacI.

Materials:

1. DNA (1 μg)

2. 10X rCutSmart Buffer (5 μL)

3. NdeI (1.0 µl)(20 units)

4. Pacl (1.0 µl)(10 units)

5. Nuclease-free Water (Bring to 50 μL)

Procedure:

1. Set up a reaction by combining the DNA, rCutSmart Buffer, NdeI, PacI, water for 50 µl total volume.

   1. For convenience, 1.0 µl is specified; adjust as needed. In general, we recommend 5–10 units of enzyme per µg DNA, and 10–20 units for genomic DNA in a 1 hour digest. Enzyme volume should not exceed 10% of the total reaction volume to preventhttps://www.neb.com/tools-and-resources/usage-guidelines/star-activity" style="text-decoration: none;"> star activity due to excess glycerol.

2. Incubate at 37°C for 5-15 minutes as both enzymes are Time-Saver qualified.

   1. DNA purified by standard miniprep procedures is cleaved at lower rates by NdeI. In this case, increasing incubation time to at least 1 hr may be necessary.

3. Heat inactivate. For heat-inactivation recommendations please refer tohttps://www.neb.com/tools-and-resources/usage-guidelines/heat-inactivation" style="text-decoration: none;"> this chart. Heat inactivation is recommended if you plan to continue to another step in the workflow without a DNA purification step.

TEV Protease Digestion

Cleave the linker and targeting peptide from the AMP after the encapsulins have been destroyed.

Materials:

1. Substrate (15 μg)

2. H20 (Bring Substrate volume to 45 μL)

3. TEV Protease Reaction Buffer-10X (5 μL)

4. TEV Protease

Procedure:

1. Combine 15 μg of substrate and H2O (if necessary) to make a 45 μl total reaction volume.

2. Add 5 μl of TEV Protease Reaction Buffer (10X) to make a 50 μl total reaction volume.

3. Add 1 μl of TEV Protease.

4. Incubate at 30°C for 1 hour or at 4°C overnight.