PROTOCOLS

Materials

• LB agar
• Antibiotic
• Petri dishes

Procedure

1. Dissolve 14.24 g of Lysogeny Broth (LB) agar in 400 ml distilled water.
2. Autoclave the LB agar solution.
3. Cool down the solution before adding antibiotics. To speed up the process, you can hold the bottle under cold tap water while stirring.
4. Supply with the appropriate antibiotics. When using 1000X antibiotic stock solutions, add 1 µl of the antibiotic stock solution per ml of LB agar.
5. Under sterile conditions, pour the LB agar in sterile Petri dishes.
6. Leave the Petri dishes near the flame with the lid partially off for approximately 5 minutes so the agar can solidify.
7. Store at 4 °C until use.

Materials

• Antibiotic of interest
• 95 % ethanol (in case of Chloramphenicol)
• MilliQ (in case of other antibiotics)

Procedure

For 1000X antibiotic stock solutions:

1. Weight X grams of the antibiotic of interest (Table 1).
Table 1. Necessary quantities for different antibiotics

Antibiotic	[Stock solution] 1000x	[Final in the medium]
Ampicillin	100 mg/ml	100 µg/ml
Kanamycin	50 mg/ml	50 µg/ml
Chloramphenicol	35 mg/ml	35 µg/ml
Terracycline	15 mg/ml	15 µg/ml

2. Dissolve in 95% ethanol (in case of Chloramphenicol) or Milli-Q water (in case of other antibiotics).

Procedure

Preparation

1. Build the electrical circuit according to the description found in the hardware assembly manual
2. Connect the Arduino Nano board to a computer using a USB cable.
3. Upload the Arduino iGEM_hardware_button.ino to the Arduino Nano using the Arduino IDE software.

Calibration

1. Take 5 capacitors with different known capacitances close to 1pF and attach the capacitor with the highest capacitance between the clamps. The second clamp will hold capacitors with varying values.
2. Open the Serial Plotter in the Arduino IDE software. Here, you see the relative output on the y-axis and the time on the x-axis.
3. Measure the relative output for each of the capacitors attached to the second clamp.
4. Use a linear regression line to plot the relative value against the difference in capacitance.
5. Note the formula of the line. This will later be used to calculate the (difference in) capacitance from the relative output.

Measurement

1. Put Electrode A (with immobilized DNA with BlcR binding site) and Electrode B (without immobilized DNA with BlcR binding site) in eppendorfs in which the fingers of the electrodes are submerged in a 1 mL solution with 4 µM BlcR in MOPS Mg buffer. Incubate the electrodes for 1 hour.
2. Open the Serial Plotter in the Arduino IDE software. Here, you see the relative output on the y-axis and the time on the x-axis.
3. Connect Electrode A and Electrode B to the circuit using the clamps. Make sure that the fingers remain submerged in the solution at all times.
4. Wait until the signal is stabilized, then baseline the signal by pressing the button. Make sure that the relative value remains around zero.
5. Add 10 µL SSA of a 15 mM stock solution to the eppendorfs and pipette up and down.
6. Check whether the relative value in the Serial Plotter has increased (with around 6 in our case).
7. Convert the relative value into the difference in capacitance with the formula of the calibration line.

In this project, we worked with the AFM instrument NanoWizard 4 from Bruker.

Procedure

1. Prepare your sample and place it on a microscope slide. Make sure your sample will not move under the AFM, by taping or UV gluing the sample to the slide for instance.
2. Choose your desired mode (for instance QI or AC mode).
3. Choose your desired probe (for instance MSNL-10B).
4. Put the piezo scanner in the probe holder.
5. Put the probe in the piezo scanner.
6. Place the piezo scanner with the probe in the AFM.
7. Put the electrode in the electrode port of the AFM.
8. Start the JPK NanoWizard control application and select your desired mode.
9. Select the right probe in the program.
10. Align the laser.
11. Make sure to decrease the Z-position so your sample won’t touch the tip and crush it.
12. Take out the AFM machine.
13. If you are imaging in liquid, add liquid to your tip by either putting a water droplet on your cantilever or put the container (with your sample) with liquid under the AFM.
14. Align the detector.
15. Now calibrate the probe in the Calibration Manager.
16. Select the to-be-used medium (air or liquid). Also don’t forget to put in the length and width of the cantilever. Select the resonance frequency.
17. Change the experiment settings to what you would expect as sample height and sample adhesion.
18. Put your sample underneath the AFM now. If you are sampling in liquid, make sure no air bubbles are present under the tip.

AC mode

1. Open AC Feedback Mode Wizard. Select Fast Scanner Directdrive and take a beginning and end frequency around your resonance peak and select your resonance frequency to be your drive frequency. Then select ‘run’. Select the frequency that is slightly lower than the resonance frequency and an amplitude that is around 60% of the maximum amplitude.
2. Start approaching.
3. Start scanning.

QI mode

1. Choose an appropriate setpoint, Z-length and speed.
2. Start scanning.

Equipment

• Spectrophotometer
• Water bath at 37°C

Materials

• Pierce BCA Protein Assay Kit [1]
• Diluent: buffer of unknown protein sample

Procedure

1. Make a 1 mL stock solution of 2 mg/mL Albumin standard (BSA).
2. Make different dilutions from the stock solution following the table. Use the buffer of your protein samples as diluent.
Table 2. Different BSA dilutions

Vial	Volume of diluent ( µL)	Volume and source of BSA ( µL)	Final BSA concentration ( µg/mL)
A	0	300 of stock	2000
B	325	325 of stock	1500
C	125	375 of stock	1000
D	175	175 of vial B dilution	750
E	325	325 of vial C dilution	500
F	325	325 of vial E dilution	250
G	325	325 of vial F dilution	125
H	400	100 of vial G dilution	25
I	400	0	0

3. Calculate the amount of working reagent needed with the following formula: (#standards + #unknowns) x (replicates) * 2 mL = volume working reagent
4. Prepare the working reagent by mixing 50 parts of Reagent A (PierceTM BCA Protein Assay Kit [1] ) and 1 part of Reagent B (PierceTM BCA Protein Assay Kit [1] ). Example: 50 mL reagent A is mixed with 1 mL reagent B
5. Pipette 0.1 mL of each standard and unknown in Eppendorf tube.
6. Add 2 mL of the working solution to all the samples.
7. Incubate the samples for 30 minutes at 37°C.
8. Cool the samples to room temperature.
9. Measure all the samples within 10 minutes with a spectrophotometer set to 562 nm.
10. Use a linear regression line to plot the absorbance against the concentration.
11. Use the formula of the regression line to calculate the protein concentrations in the unknown samples.

Equipment

• Large tabletop centrifuge with temperature control OR:
• Cold room (4°C)

Materials

• Dialysis cassette OR:
• Protein concentrator centrifuge tube
• Buffer you wish to exchange to

Procedure

Buffer exchange by concentrator tube

Need: large tabletop centrifuge with temperature control, protein concentrator tube, buffer.

1. Make sure your buffer is cooled to 4°C.
2. Set the centrifuge to cool to 4°C.
3. Add the protein solution to the top section of the concentrator (if you have too much solution, spin down and then add more solution).
4. Centrifuge according to the user manual of your concentrator tubes (15 minutes at 4,000 x g for ThermoFisher Pierce 10 MWCO concentrators).
5. Check whether a significant volume has flown through, remove the filtrate from the bottom, add a buffer to fill the top compartment, and use a pipette to properly mix the protein solution at the bottom of the filter. Centrifuge again. Perform this cycle 10 times.
6. Finally, transfer the protein solution from the top compartment to a clean tube.

Buffer exchange by dialysis cassette

Need: dialysis cassette, cold room (4°C), buffer.

1. Prepare roughly 2 L of the buffer you wish to transfer your protein to for every 10 mL of protein solution, and cool the buffer to 4°C before you start.
2. Move to the cold room.
3. Place a stirrer magnet into a large beaker (≥1 L) and place on a magnetic stirrer plate. Add 1 L of buffer.
4. Drop the dialysis cassette into the buffer and let it absorb for 1 minute.
5. Draw up to 12 mL of protein solution in a syringe.
6. Insert the syringe needle in one of the channels on the corners of the cassette. Slowly inject the protein solution, occasionally drawing air out of the cassette, until the cassette is full and there are no big air bubbles.
7. Attach a float buoy to the edge of the cassette (preferably the long edge) and make sure it is well centered. Drop the cassette with the buoy into the beaker with the buffer. If the bottom of the cassette is very close to the stirrer magnet, add more buffer.
8. Turn on the magnetic stirrer, at a speed at which the cassette is rotating slowly.
9. Leave to rotate ≥7 hours or overnight, then replace the buffer with another liter.
10. Leave to rotate ≥7 hours or overnight.
11. Take a syringe and draw 10 mL of air
12. Insert the syringe needle in one of the corner channels of the cassette. Draw the protein solution into the syringe, occasionally injecting air into the cassette to neutralize pressure.
13. Transfer the protein solution from the syringe to a clean tube. If precipitation is visible, centrifuge at max speed for 5 minutes and transfer the supernatant to a clean tube.

Protocol taken from Pan et al. [2]

Procedure

Gel preparation

1. In a Duran bottle:
Table 3. EMSA gel preparation

Component	Volume
TAE 10x	4 mL
30% acrylamide-bis	2 mL
Distilled water	9 mL

2. Let the mix cool down at room temperature for 15 minutes.
3. Consequently add:
Table 4. EMSA gel preparation

Component	Volume
TEMED	15 uL
10% APS	75 uL

4. Cast the gel and let it polymerize in the gel cassette for at least 1 hour. (NB be prepared for casting when adding TEMED and APS as the gel tends to solidify soon after).

Binding buffer preparation

1. In an Eppendorf tube, prepare a binding buffer working solution for 10 samples.
Table 5. EMSA binding buffer preparation

Component	Volume	Final concentration
Tris-Cl, pH 8 [20 mM]	20 µL	2 mM
EDTA [2.5 mM]	16 µL	0.2 mM
DTT [20 mM]	20 µL	0.2 mM
Potassium Glutamate [120 mM]	20 µL	12 mM
BSA [1µg/µL]	40 µL	4 µg/µL
Glycerol 20%	20 µL	2 %

2. Let the mix cool down at room temperature for 15 mins.

Sample preparation

1. In Eppendorf/PCR tubes prepare the samples as follows:
Table 6. EMSA sample preparation

Component	Sample 1	Sample 2	Sample 3	Sample 4	Sample 5
Cy3 DNA [100 nM]	4 µL	4 µL	4 µL	4 µL	4 µL
BlcR [2.9 µM]	0 µL	1.6 µL	2.6 µL	3.6 µL	4.6 µL
Binding Buffer	13.6 µL	13.6 µL	13.6 µL	13.6 µL	13.6 µL
Nuclease free water	5.4 µL	3.8 µL	2.8 µL	1.8 µL	0.8 µL
Total Volume	23 µL	23 µL	23 µL	23 µL	23 µL

*The protein concentration in the sample is accordingly 0.78 uM, 1.1 uM, 1.4 uM and 1.6 uM. This allows for 25%, 50%, 75% and 100% binding of BlcR to DNA.

2. Incubate the samples for 15 minutes in the dark at room temperature. (NB It is very important that the samples are incubated in the dark as Cy3 DNA is light sensitive)
3. Consequently add:

Gel running

1. Fill the gel cassette with running buffer (TAE 1x).
2. Load the samples in the gel.
3. Run the gel at 90V for 40 minutes.

Gel visualization

1. Visualize the gel with the gel doc.

Equipment

• GelDoc imaging system

Materials

• TAE buffer
• Agarose
• SYBR safe

Procedure

1. Add 1.2 g of agarose to 100 mL of TEA buffer.
2. Heat in microwave until the agarose dissolved.
3. Cool down until the bottle is hand warm.
4. Pour the agarose gel into a mall.
5. Add 5 µL of SYBR safe (60.000x) into gel and stirr.
6. Put the camb inside the gel.
7. Let the gel stool for around 40 minutes.
8. Add 2 µL of DNA loading dye (6x) to 10 µL of PCR product.
9. Load 10 µL of your sample next to a DNA ladder.
10. Run gel at 70V for 60-80 minutes.
11. Visualize gel with trans UV.

Procedure

1. Prepare a glucose stock solution of 2.5 mM in the buffer you want to measure the glucose concentration in (in our case HEPES).
2. Make the dilutions described in Table 7.
Table 7. Dilutions for glucose determination assay

Glucose concentration (mM)	HEPES buffer (µL)	Glucose 2.5 mM stock solution (µL)
0	1000	0 of stock
0.5	800	200
0.75	700	300
1.1	550	440
1.5	400	600
2	200	800
2.5	0	1000

3. Vortex all eppendorfs with the dilutions.
4. Set the spectrophotometer to 505 nm.
5. Transfer 50 µL from each dilution to a new eppendorf. Do this twice to measure in duplo.
6. Transfer 50 µL of your sample to a new eppendorf. Do this three times to measure in triplo.
7. Add 1 mL of glucose reagentia to each eppendorf.
8. Vortex all eppendorfs.
9. Incubate the eppendorfs in a water bath at 37 degrees Celsius for 10 minutes.
10. Cool the samples to room temperature.
11. Transfer the solution in all eppendorfs in a cuvette.
12. Measure the absorbance of all solutions at 505 nm.
13. Use a linear regression line to plot the absorbance against the concentration.
14. Use the formula of the regression line to calculate the glucose concentrations in the unknown samples.

Equipment

• -80 °C freezer
• Incubator

Materials

• 80% glycerol
• Milli-Q
• Inoculated culture

Procedure

1. Prepare a 80% (v/v) glycerol solution in Milli-Q and sterilize by autoclaving.
2. Inoculate 10 mL LB liquid bacterial culture containing the appropriate antibiotic. Incubate at 37 °C overnight, shaking at 180 rpm.
3. Add 250 µL of the 80% glycerol solution to a sterile cryotube.
4. Add 750 µL of the liquid bacterial culture to the tube and mix well.
5. Store at -80 °C.

Equipment

• Large tabletop centrifuge with temperature control
• Cold room (4°C)
• Falcon tube rotator

Materials

• His-tag affinity beads in resin (we use ThermoFisher HisPur Ni-NTA resin)
• 20 mL gravity column

Procedure

Washing Buffer (pH 7.5)

Elution Buffer (pH 7.5)

His-tag affinity chromatography

Need: Clarified lysate, His-tag affinity beads resin (nickel or cobalt), 20 mL gravity column, washing buffer, elution buffer (see recipes), cold room, large table centrifuge, 50 mL tubes.

Note: all following steps are to be performed at 4°C.

1. Resuspend the bead resin.
2. Add beads to a 50 mL falcon tube. Use roughly 1 mL of suspended resin per 15 mL of clarified lysate. Spin down for 1 min at 3220 g and discard the supernatant.
3. Resuspend in 20 mL washing buffer, spin down for 1 min at 3220 g and discard the supernatant. Do this three times.
4. Add the clarified lysate to the tube with the beads. Resuspend the beads and incubate for 1 hr, rotating slowly, at 4°C.
5. Load the bead suspension onto a gravity column and let the liquid flow through, collecting it in a clean tube labelled “flow through”, and store in the fridge.
6. Resuspend the beads in a 20 mL washing buffer and load the mixture onto a gravity column. Let the buffer flow through and collect it in a clean tube and store in the fridge.
7. Close the column tap. Add 12 mL elution buffer and resuspend the beads. Leave it to bind for 5 minutes.
8. Without adding new elution buffer, resuspend the beads again and leave to bind for 5 minutes again. Do this three times.
9. Open the tap of the gravity column and collect the elution in a clean tube. Store in the fridge.

You should have collected four products: the lysate flow-through, the wash, and the elution, as well as a sample of the clarified lysate. If you used multiple columns, combine the products of each into one tube. Store all in the fridge. At this point, run an SDS-PAGE with samples of each fraction to check the purification process.

Regenerating HisPur Ni-NTA beads

Note: beads can be reused up to 4 times without affecting protein yield or purity.

1. Prepare the regeneration buffer: 20 mM MES, 100 mM NaCl, pH 5.0.
2. Resuspend used beads in regeneration buffer and add to a clean gravity flow column.
3. Wash beads with two column volumes regeneration buffer.
4. Wash beads with two column volumes Milli-Q water.
5. Resuspend the beads in one resin bed volume of 20% ethanol in XXXXXXX, transfer to a falcon tube (write the number of times the beads have been reused on it) and store at 4°C.

In order to immobilize DNA on the electrodes and verify the binding of DNA, BlcR on the DNA and verify the BlcR unbinding caused by SSA, the following subprotocols were performed.

Procedure

Buffer preparation

1. To make the MOPS buffer, add:
• 20 mM MOPS
• 30 mM sodium sulfate (Na₂SO₄)
2. in Milli- Q. Adjust the pH to 7 with H₂SO₄ and NaOH.
3. To make the MOPS Mg buffer, add:
• 2.5 mM Magnesium sulfate (MgSO₄)
• 20 mM MOPS
• 30 mM sodium sulfate (Na₂SO₄)
4. in Milli- Q. Adjust the pH to 7 with H₂SO₄ and NaOH.

Preparation of the IDE

Before the DNA can be incubated on the electrodes, it is important that the electrodes are clean. The cleaning of the electrodes is done with sonication. The following protocol, based on the protocol in the paper of Georgas et al. (2022) [3] is used.

1. Take an IDE very carefully with a pincet and try to only touch the plastic. Put the IDE in a clean p=Petri dish.
2. Take a beaker glass and put your IDE in it. Cover the electrode with a generous amount of pure isopropanol.
3. Turn on the sonication bath for 5 minutes.
4. If it finishes, take out the electrode carefully and place it in a second Petri dish.
5. Carefully wash both sides of the electrode with Milli-Q.

Preparation of the DNA

The freeze dried ordered oligonucleotides were first resuspended in MOPS buffer. Stock solutions of 100 µM DNA were made. The thiol modification on one of the single strands induces disulfide bridges between the DNA strands. This prevents the thiol to bind to the gold electrodes and therefore, the thiol modified single strands need to be reduced before DNA immobilization.

The reduction of the DNA is done with the reducing agent dithiothreitol (DTT). The following protocol has been used to incubate one electrode (multiply if multiple electrodes are incubating at the same time). This protocol is based on the protocol of Base Pair Biotechnologies [4] and BIONEER [5] .

1. Make 100 mM stock solution of DTT in MOPS (100 µL) (10 µL DTT, 90 µL MOPS).
2. Make 11 µL solution with:
• 100 µM DNA → 10 µL of 100 µM stock solution.
• 10 mM DTT → 1 µL of 100 mM stock solution.
3. Vortex the eppendorf and incubate at room temperature for 15 minutes.
4. Add 11 µL ethyl acetate to the eppendorf.
5. Vortex the eppendorf shortly.
6. Centrifuge the eppendorf shortly for around 10 seconds.
7. Two layers should now be visible. Remove the upper layer and continue with the bottom layer.
8. Repeat steps 4-7 three more times.

Immobilization of the first single strand

It is of utmost importance that the reduced DNA is directly incubated on the electrode to avoid oxidation again. Perform the following steps directly after the last step of step 3.

1. Add 990 µL of MOPS buffer to the reduced DNA.
2. Directly the electrode in this eppendorf with 1 mL solution. Make sure that all fingers of the electrode are covered.
3. Incubate the electrode in this bath overnight or for 6-9 hours.

Washing

After incubation, the electrode needs to be washed very extensively to remove all unbound DNA. The washing is done as follows.

1. Take the electrode out of the eppendorf and hold it with your tweezers under an angle.
2. Place a piece of tissue under the electrode.
3. Pipet very carefully 1 mL of MOPS buffer from the top of the electrode and let the buffer run over the whole electrode. This way, a very simple flow cell is created.
4. Now turn over the electrode to wash the other side. Wash this side again with 1 mL of MOPS buffer.

Mercaptohexanol

To prevent the rest of the molecules from sticking to the electrode’s surface, mercaptohexanol (MCH) is used. MCH, which also contains sulfur, forms a short protective layer on the gold.

1. Make a stock solution of 1 mM MCH in MOPS buffer.
2. After the previous washing step, incubate the electrode in a bath of 1 mL 1 mM MCH.

Washing

After incubation, the electrode needs to be washed very extensively to remove all unbound DNA. The washing is done as follows.

1. Take the electrode out of the eppendorf and hold it with your tweezers under an angle.
2. Place a piece of tissue under the electrode.
3. Pipet very carefully 1 mL of MOPS buffer from the top of the electrode and let the buffer run over the whole electrode. This way, a very simple flow cell is created.
4. Now turn over the electrode to wash the other side. Wash this side again with 1 mL of MOPS buffer.

Immobilization of the second single strand

It is of utmost importance that the reduced DNA is directly incubated on the electrode to avoid oxidation again. Perform the following steps directly after the last step of step 3.

1. Add 990 µL of MOPS buffer to the reduced DNA.
2. Directly the electrode in this eppendorf with 1 mL solution. Make sure that all fingers of the electrode are covered.
3. Incubate the electrode in this bath overnight or for 6-9 hours.

Washing

After incubation, the electrode needs to be washed very extensively to remove all unbound DNA. The washing is done as follows.

1. Take the electrode out of the eppendorf and hold it with your tweezers under an angle.
2. Place a piece of tissue under the electrode.
3. Pipet very carefully 1 mL of MOPS buffer from the top of the electrode and let the buffer run over the whole electrode. This way, a very simple flow cell is created.
4. Now turn over the electrode to wash the other side. Wash this side again with 1 mL of MOPS buffer.

Addition of BlcR

It is of utmost importance that the Mg MOPS buffer will be used in the following (washing) steps. The magnesium allows the BlcR to bind more easily.

1. Make a stock solution of 1 mL 2.45 µM BlcR in Mg MOPS.
2. Incubate the electrode in this bath at room temperature for 1 hour.

Addition of SSA or GHB

To release the BlcR again, SSA or GHB can be used.

1. Prepare a 15 mM stock solution of SSA/GHB.
2. Take 10 µL of the SSA or GHB stock solution and add it to the BlcR bath.
3. Carefully pipet up and down to homogenize the solution.
4. Incubate this solution for 15 minutes.

Equipment

• Incubator

Materials

• Antibiotic
• LB medium

Procedure

1. Add 10 mL LB media to a tube.
2. Add the antibiotic needed for selection.
3. Pick a single colony using either a sterile pipet tip, a sterile toothpick or a sterilized inoculation loop.
4. Shake the tip, toothpick or inoculation loop containing the bacterial colony in the LB media.
5. Incubate tubes overnight in an incubator at 37 °C shaking at 180 rpm.

Equipment

• Using the MicroCal VP-ITC

Procedure

Starting up

1. Switch on the VP-ITC.
2. Switch on the computer and open the VP-ITC program.
3. Create a new protocol and set the required temperature (will take a while).
4. Switch on the heating block and set the temperature at 2°C below the desired experiment temperature.
5. While you wait, prepare sample solutions.
6. Clean the cell and syringe using storage buffer.

Cleaning the cell

1. Empty the cell.
2. Wash thoroughly with 2% SDS three times using the filling needle.
3. Wash thoroughly with the buffer your sample is in (or distilled water, if cleaning for shutdown) three times, using the filling needle.

Cleaning the syringe

1. Empty the syringe into a waste tube by opening the fill port.
2. Clean the outside with water and wipe down gently with a tissue.
3. Put the syringe into a tube of 2% SDS.
4. Open the fill port, draw some solution, close the fill port.
5. Purge & refill three times.
6. Empty by opening the fill port.
7. Repeat from step 2, using storage buffer.

Running an experiment

1. Make sure the temperature of the ITC and the heating block are set correctly.
2. Degas your samples (keep an eye on the protein samples; carefully open the valve).
3. Fill the cell:
1. Make sure it is completely empty by drawing from the bottom of the cell.
2. Clean the filling needle with buffer and replace the syringe with a clean one.
3. Draw the sample, without drawing bubbles.
4. Put the needle slightly above the bottom of the cell and fill until liquid rises above the cell port.
5. Tilt the needle and give some small bursts of the syringe to dislodge any bubbles stuck in the cell.
6. Remove any liquid above the rim of the fill port (use cotton swab for droplets).
4. Fill the syringe:
1. Make sure the syringe is empty and wipe the outside.
2. Put the syringe into your degassed sample.
3. Open the fill port and draw the sample into the syringe; don’t draw any air.
4. Close the fill port.
5. Purge & refill three times.
5. Take the syringe and clean the outside by rinsing with water and wiping it down.
6. Gently insert the syringe into the cell, make sure it clicks shut.
7. Setting up the protocol:
1. Set the injection regime: number, volume, time, and interval.
2. Set the first injection to 2 µL.
3. Set the concentrations of the samples.
4. Set the reference power (If unknown, set to 15).
5. Set the stirring speed.

Shutting down

1. Clean the cell and syringe using 2% SDS and distilled water, and leave them filled with distilled water.
2. Put the lid on the cell.
3. Switch off the computer.
4. Switch off the pump/heating block.
5. Switch off the VP-ITC.

Materials

• T4 DNA ligase buffer
• T4 Kinase
• T4 DNA ligase
• DpnI

Procedure

1. Add the following components in a PCR tube:
Table 10. KLD reaction components

Component	Volume ( µL)	Final Concentration
T4 DNA ligase buffer	1 µL	10x
T4 PNK	1 µL	0.1 U/µL
T4 DNA ligase	1 µL	0.4 U/µL
Dpnl	1 µL	0.2 U/µL
PCR product	1 µL
MilliQ	5 µL

2. Incubate for 1 hour at room temperature.
3. Move to transformation protocol to insert the plasmid inside E. coli.

Materials

• Lysogeny Broth (LB) powder

Procedure

1. Dissolve Lysogeny Broth (LB) powder in distilled water according to manufacturer instructions.
2. Autoclave the LB solution.
3. Store at room temperature until use.

Protocol adapted from PureYield™ Plasmid Miniprep [6]

Equipment

• Centrifuge

Materials

• Miniprep kit from promega [6]
• Inoculated culture

Procedure

For small volumes

Prepare Lysate

1. Add 2 mL of bacterial culture to a 2 mL microcentrifuge tube (for higher yields and purity use the laternative procol below to process up to 3 mL).
2. Add 100 µL of Cell Lysis Buffer, and mix by inverting the tube 6 times.
3. Add 350 µL of cold (4-8°C) Neutralization Solution, and mix thoroughly by inverting.
4. Centrifuge at maximum speed in a microcentrifuge for 3 minutes.
5. Transfer the supernatant (~900 µL) to a PureYield ™ Minicolumn without disturbing the cell debris pellet.
6. Place the minicolumn into a Collection Tube, and centrifuge at maximum speed in a microcentrifuge for 15 s.
7. Discard the flowthrough, and place the minicolumn into the same Collection Tube.

Wash

1. Add 200 µL of Endotoxin Removal Wash (ERB) to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 15 s.
2. Add 400 µL of Column Wash Solution (CWC) to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 30 s. (repeat twice if necessary).

Elute

1. Transfer the minicolumn to a clean 1.5 mL microcentrifuge tuce, then add 40 µL of heated nuclease-free water directly to the minicolumn matrix. Incubate for 1 minute at room temperature.
2. Centrifuge for 15 s to elute the plasmid DNA. Cap the microcentrifuge tube, and sotre eluted plasmid DNA at -20°C.

For larger volumes

1. Centrifuge 10 ml of bacterial culture for 5 min at maximum speed in an ultracentrifuge. Discard the supernatant.
2. Add 600 µL of water to the cell pelle, resuspend completely.
3. Proceed to step 2 of the standard protocol above.

Equipment

• NanoDrop UV-Vis Spectophotometer

Procedure

1. Press the button dsDNA to measure the concentration of double stranded DNA in your samples.
2. Clean the measurement surface with kimwipes and Milli-Q water.
3. Use 1 µL of the same buffer the sample is dissolved in to set the blank.
4. Use 1 µL of sample to measure its concentration. If you have multiple samples, clean the measurement surface in between measurements with kimwipes.

Equipment

• GeneJET PCR Purification kit
• PCR construct

Procedure

1. Add a 1:1 volume of Binding Buffer to the completed PCR mixture Mix thoroughly.
2. Transfer up to 800 µL of the solution from step 1 to the GeneJET purification column.
3. Centrifuge for 30-60 s at maximum speed. Discard the flow-through.

Notes. If the total volume exceeds 800 µL, the solution can be added to the column in stages. After the addition of 800 µL of solution, centrifuge the column for 30-60 s and discard flowthrough. Repeat until the entire solution has been added to the column membrane.

4. Add 700 µL of Wash Buffer to the GeneJET purification column.
5. Centrifuge for 30-60 s. Discard the flow-through.
6. Place the purification column back into the collection tube.
7. Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove any residual wash buffer.
8. Place the purification column into a clean tube.
9. Add 35 µL of preheated milliQ to the GeneJET purification column membrane and centrifuge for 1 min.
10. Discard the GeneJET purification column and store the purified DNA at -20 °C.

Protocol adapted from ThermoFisher scientific (Phusion™ High–Fidelity PCR kit) [7] .

Equipment

• PCR machine

Materials

• Phusion™ High–Fidelity PCR kit [7]
• Template DNA (1 ng)
• Forward and reverse primers (10 uM)

Procedure

1. Add the following components in a PCR tube:
Table 11. Necessary quantities for Phusion PCR reaction

Component	Volume (20 µL)	Final Concentration
5x Phusion buffer HF	4 µL	1x
10 mM dNTPs	0.4 µL	200 µM each
Forward Primer 10 µM	1 µL	0.5 µM
Reverse Primer 10 µM	1 µL	0.5 µM
Template DNA (1 ng)	X µL	1 ng
Phusion DNA polymerase	0.2 µL	0.02 U/µL
Milli-Q	Add to 20 µL
Total	20 µL

2. Introduce the PCR tubes in the thermocycle and use the following PCR protocol:
Table 12. Phusion PCR thermocycle

Cycle Steps	Temperature (°C)	Time	#cycles
Initial Denaturation	98	30s	1
Denaturation	98	5-10s	25-35
Annealing	X (according to Tm)	10-30s
Extention	72	15-30 s/kb
Final Extention	72	5-10 min	1
Hold	4L	∞	1

3. Analyze PCR products with gel electrophoresis.
4. For site directed mutagenesis move on to KLD protocol.

Protocol adapted from Thermo scientific (Q5 High–Fidelity PCR kit)

Equipment

• PCR machine

Materials

• Q5 PCR kit
• Template DNA (1 ng)
• Forward and reverse primers (10 µM)

Procedure

1. Add the following components in a PCR tube:
Table 13. Necessary quantities for Q5 PCR reaction

Component	Volume (25 µL)	Final Concentration
5x Q5 Reaction Buffer	5 µL	1x
10 mM dNTPs	0.5 µL	200 µM each
Forward Primer 10 µM	1.25 µL	0.5 µM
Reverse Primer 10 µM	1.25 µL	0.5 µM
Template DNA (1 ng)	0.5 µL	1 ng
Phusion DNA polymerase	0.25 µL	0.02 U/µL
Milli-Q	Add to 20 µL
Total	25 µL

2. Introduce the PCR tubes in the thermocycle and use the following PCR protocol:
Table 14. Q5 PCR thermocycle

Cycle Steps	Temperature (°C)	Time	#cycles
Initial Denaturation	98	30s	1
Denaturation	98	5-10s	25-35
Annealing	X (according to Tm)	10-30s
Extention	72	15-30 s/kb
Final Extention	72	5-10 min	1
Hold	4L	∞	1

Equipment

• Large tabletop centrifuge with temperature control
• Large-capacity centrifuge (with 250 mL, 500 mL or 1 L tubes)
• Ultracentrifuge (at least 20.000 rpm)
• Probe-type sonicator OR French press cell disruptor

Materials

• BL21(DE3) competent cells and pET-11a-T7tag-6xHis-TEV-BlcR plasmid OR:
• Glycerol stock of BL21(DE3) carrying pET-11a-T7tag-6xHis-TEV-BlcR
• LA-Amp plate
• LB-Amp
• 5 L shake flask
• IPTG
• PBS

Procedure

Washing Buffer (pH 7.5)

Lysing Buffer

1. Take 100 mL of the washing buffer and add a protease inhibitor cocktail.

Transformation (if necessary)

Need: BL21(DE3) competent cells, pET-11a-T7tag-6xHis-TEV-BlcR plasmid, LA-Amp plate.

Keep competent cells on ice at all times and put the tube back at -80°C as soon as you have taken the amount you need.

1. Set one heating block to 42°C and another to 37°C.
2. Let SOC medium come to room temperature.
3. Add 1-10 ng pET-11a-T7tag-6xHis-TEV-BlcR to 20 µL BL21(DE3) competent cells, keeping it on ice, and mix gently by flicking the tube.
4. Incubate on ice for 30 minutes.
5. Heat shock at 42°C for exactly 10 seconds.
6. Rest on ice for 5 minutes.
7. Add 380 µL room temperature SOC medium.
8. Incubate at 37°C for 1 hour, shaking at 300 rpm.
9. Plate 50 µL of the culture on LA-Amp.
10. Make a 10x dilution of the culture in SOC medium and plate 50 µL on LA-Amp.
11. Seal and incubate at 37°C overnight.

Reviving glycerol stock (if necessary)

Need: sterile toothpick, LA-Amp plate.

Do not let the glycerol stock thaw; do the following in the room with the freezer and put the tube back as soon as you are done:

1. Take the E. coli BL21(DE3) glycerol stock containing the pET-11a-T7tag-6xHis-TEV-BlcR plasmid from the -80°C freezer.
2. Using a toothpick or any other sterile inoculating needle, scrape a small amount of culture from the glycerol stock. The clump of scraped material at the end of the toothpick should be barely visible. Put the glycerol stock back at -80°C.
3. Use the toothpick and a clean blunt streaking needle to streak the culture on an LA-Amp plate.
4. Seal and incubate at 37°C for >7 hours or overnight.

Inoculation

Need: toothpick, 50 mL tube, LB, ampicillin, sterile 5 L shake flask, IPTG.

1. Put 10 mL LB into a 50 mL tube and add ampicillin.
2. Poke a single colony from the plate of transformants with a toothpick and drop it into the tube with LB-Amp.
3. Incubate, shaking at 180 rpm, at 37°C overnight (but not much longer). Make sure the cap is only loosely on the tube.

Optional: to make a glycerol stock at this point, take 500 µL of the overnight culture and add 500 µL 50% glycerol. Store at -80°C.

Note: BL21(DE3) is not fit for long term storage. Do not use this stock for plasmid cultivation.

Cultivation

Need: overnight culture, LB, ampicillin, sterile 5 L shake flask, 1M IPTG stock.

Note: from this point the protocol can be multiplied to produce more protein.

1. Prepare 1 L of LB-Amp and add to a sterile 5 L erlenmeyer flask.
2. Pour in the 10 mL of overnight culture (divide equally if using multiple flasks).
3. Incubate, shaking at 180 rpm, at 37°C, until OD has reached 0.6-0.8 (check after 2 or 3 hours). Cover the opening of the flask with aluminium foil, do not seal.
4. When OD has reached 0.6-0.8, put the culture on ice for 30 minutes. Let the incubator cool to 18°C.
5. Add 1M IPTG stock to a final concentration of 1 mM (1:1000).
6. Incubate shaking at 18°C overnight.

Culture harvesting and extraction

Need: cultivation culture, large capacity centrifuge, large table centrifuge, PBS, lysis buffer (see recipe), probe type sonicator, 50 mL tubes.

1. Keep the overnight culture on ice after it is done incubating.
2. Cool the large capacity centrifuge and the large table centrifuge to 4°C.
3. Transfer the culture to 500 mL or 1L centrifugation tubes. Make sure they weigh exactly the same and document the weight.
4. Centrifuge at 4°C, 4000 rpm, 20 minutes.
5. Discard the supernatant and weigh the pellet.
6. Resuspend the pellet in 30 mL 1X PBS and transfer to a 50 mL falcon tube.
7. Centrifuge at 4°C, 4000 rpm, 20 minutes.
8. Discard the supernatant.

(You can pause at this point and store the pellet at -80°C, or continue)

Note: all following steps are done at 4°C or on ice.

9. Cool the ultracentrifuge to 4°C.
10. Thoroughly resuspend the pellet in 40 mL cold lysis buffer
• Sonicate (on ice) using the 422A tip at 40% amplitude, 10s/10s pulse, for 10 min (will take 20 min). OR:
• Add a knife-tip of DNAse and lyse the culture in a french press type cell disruptor.
11. Transfer the lysate to ultracentrifuge tubes, making sure they weigh exactly the same.
12. Ultracentrifuge at 20.000 rpm or if possible 40.000 rpm for 30 min.
13. Transfer the supernatant (clarified lysate) to a 50 mL tube as soon as the centrifuge is done. Discard the pellet (remove by cleaning the centrifuge tube).
14. Take a 30 µL sample of the clarified lysate. Store in the fridge.

(You can pause at this point and store the clarified lysate at -80°C, or immediately continue with purification)

Protocol adapted from PUREfrex Genefrontier [8] .

Equipment

• Fluorescence plate reader

Materials

• PUREfrex2.0 [8]
• Template DNA

The amounts given are for a 20 µL reaction. For scaling up the reaction, adjust the volume of reagents accordingly.

Procedure

1. Thaw Solution I by incubation at room temperature or 37 °C for 1 minute for completely dissolving, and then cool on ice.
2. Thaw Solution II and III on ice.
3. Mix Solution I, II, and III respectively by vortex and centrifuge briefly to collect each solution at the bottom.
4. Assemble the reaction mixture in a tube as follows. Add the template DNA to 1 - 3 ng/µL per 1 kb.
Table 16. Reaction components

Component	Volume
Water	7-X μL
Solution I	10 μL
Solution II	1 μL
Solution III	2 μL
Template DNA *1	X μL
Total	20 μL

5. Incubate the samples at 37°C.
6. Measure the fluorescence at excitation 485 nm and emission 528 nm after six hours with a plate reader.

Protocol adapted from PUREfrex Genefrontier [8] .

Equipment

• Typhoon laser-scanner platform Cytiva Life Sciences

Materials

• PUREfrex2.0 [8]
• Template DNA
• GreenLys
• 4x SDS sample buffer
• 4 - 10 % Bis-Tris SDS PAGE
• MES running buffer

The amounts given are for a 20 µL reaction. For scaling up the reaction, adjust the volume of reagents accordingly.

Procedure

1. Thaw Solution I by incubation at room temperature or 37 °C for 1 minute for completely dissolving, and then cool on ice.
2. Thaw Solution II and III on ice.
3. Mix Solution I, II, and III respectively by vortex and centrifuge briefly to collect each solution at the bottom.
4. Assemble the reaction mixture in a tube as follows. Add the template DNA to 1 - 3 ng/µL per 1 kb.
Table 17. Reaction components

Component	Volume
Water	7-X μL
Solution I	10 μL
Solution II	1 μL
Solution III	2 μL
Template DNA *1	X μL
GreenLys	0.5 μL
Total	20 μL

5. Incubate the samples at 37°C.
6. Add 1 μL of RNAse A and incubate at 37°C for 1 hour.
7. Add sample buffer in a ration of 1:4 to your sample and incubate for 5 minutes at 95°C.
8. Load the samples on gel next to a protein ladder.
9. Visualize the gel with Typhoon laser-scanner, use the settings:

Equipment

• Oven at 37°C

Materials

• LB plate with antibiotic
• Glycerol stock

Procedure

Do not let the glycerol stock thaw; do the following in the room with the freezer and put the tube back as soon as you’re done:

1. Take the glycerol stock from the -80°C freezer.
2. Using a toothpick or any other sterile inoculating needle, scrape a small amount of culture from the glycerol stock. The clump of scraped material at the end of the toothpick should be barely visible. Put the glycerol stock back at -80°C.
3. Use the toothpick and a clean blunt streaking needle to streak the culture on an LB plate with the right antibiotic.
4. Seal and incubate at 37°C overnight.

Equipment

• PA-gel electrophoresis container
• Electrophoresis power source

Materials

• SDS-PAGE gel (self-made or precast, we use ThermoFisher NuPage 4%)
• SDS-PAGE 4X sample buffer (self-made or premade product)
• Protein ladder (pre-stained or not)
• SDS-PAGE running buffer
• Coomassie Blue protein staining solution of any kind

Procedure

1. Take 15 or 30 µL samples of the protein solutions you want to run.
2. Add 5 or 10 µL 4X SDS-PAGE sample buffer to each sample (in fume hood if the sample buffer contains mercaptoethanol).
3. Incubate the samples at 90°C for 10 minutes.
4. Load a protein ladder (volume suggested in the user manual of the ladder), and 10 or 20 µL of each sample (depending on slot size) in each slot. (If you don’t know how concentrated a protein sample will be, you can run different quantities of that sample in different slots, like 10 µL, 2 µL 0.5 µL)
5. Run the gel at 200 V for 25 minutes.
6. Stain the gel with Coomassie Blue for 1 hour, shaking gently (RT) or overnight (4°C).
7. Unstain in distilled water, shaking gently, for 2 hours total, replacing the water every 30 minutes.
8. Visualize the gel using a gel scanner on Trans UV mode, or on a white plate using visible light. Alternatively, use a flatbed scanner or a regular camera to take a clear picture of the gel.

Materials

• Sequencing labels
• Sequencing primer (5-10 uM)
• Purified DNA template

Procedure

1. Use 1.5 mL Eppendorf Tube.
2. Add 5 µL of miniprep product to 5 µL (> 50 ng/uL) of T7 promoter (5 - 10 µM).
3. Label the tubes using the specific labels provided by the sequencing company.

Equipment

• HiprepTM 16/60 sephcercylTM

Procedure

Preparation buffer in protein solution

1. Make the storage buffer: 50 mM HEPES, 10 % glycerol, 300 mM NaCl, pH 7.2.
2. Use a concentrator tube to concentrate the protein to a volume of 2.2 mL.
3. Pour protein solution in a 30,000 MWCO concentrator tube.
4. Centrifuge at max speed for 30 min.
5. If the protein solution is around 2.2 mL stop, if not centrifuge at max speed for 10 min until the right volume is reached.
6. Pipet the protein solution into an 2 mL eppendorf tube.
7. Centrifuge at max speed for 10 min to get rid of all the precipitation.
8. Transfer supernatant to a clean eppendorf tube.

Preparation Akta

1. Attach the gel filtration column onto an Akta purification machine.
2. Set the max pressure to the max pressure of the column, in our case 0.15 Megapascal.
3. Clean the column by washing with 2x column volume with MilliQ.
4. Equilibrate the column by washing with 1x column volume with the protein storage buffer.

Gel filtration

1. Set pre column pressure to 0.5 Mpa.
2. Set system pressure to 0.15 Mpa.
3. Set flow to 0.5 mL/min.
4. Set fractioning to 1 mL.
5. Detect at 280 nm.
6. Flush 8 mL injection loop with MilliQ to get rid of air bubbles in the loop.
7. Flush 8 mL injection loop with buffer.
8. Make sure you remove all the bubbles in the syringe with the 2.2 mL of your sample.
9. Inject your sample into the injection loope.

Check elution pattern. Peaks at 280 nm present proteins. Choose the fractions that correspond to the peak and put them on SDS PAGE to check which fraction contains your protein of interest.

Adapted from the ScreenTape user guide [9] and the 2200 TapeStation user guide [10]

Equipment

• Agilent 2200 TapeStation System (or other TapeStation system)
• Vortex mixer
• PCR tube strip centrifuge

Materials

• Agilent D5000 High Sensitivity ScreenTape kit (ScreenTape and sample buffer)
• Loading tips (5067- 5152 or 5067- 5153)
• Optical Tube 8x Strip (401428) and Optical Cap 8x Strip (401425)

Procedure

1. Let the D5000 High Sensitivity sample buffer and the ScreenTape come to room temperature for 30 minutes before use.
2. Prepare your samples in whatever tubes you prefer. Samples must be at least 2 µL. Note you can only run an even number of samples on the TapeStation.
3. Prepare and label the number of optical PCR tubes you need, keeping them in a strip. Note that the number of tubes in a strip should also be even (e.g. if you want to run 10 samples, use 8+2 tubes rather than 5+5).
4. Vortex the sample buffer well and tap the liquid down.
5. Add 2 µL of sample buffer to each tube. Pipette against the side/bottom of the tube and make sure no liquid stays inside/on the tip.
6. Add 2 µL of DNA sample to each tube. Pipette into the buffer, pipette up and down a few times and make sure no liquid stays inside/on the tip.
7. Close the tubes very well using a cap strip, and label the tops.
8. Carefully spin down the samples using a PCR tube strip centrifuge. After this, be careful when handling the tubes to make sure the samples stay in the bottom of the tubes.
9. Turn on the TapeStation system, open the TapeStation controller software, and let the system initialize.
10. Mount the sample holder and the tip holder in the TapeStation (if not already done).
11. Fill the tip holder with loading tips using a multi-tip pipette.
12. Remove the ScreenTape from its packaging, check the columns for air bubbles, and tap or flick to dislodge any.
13. Place the ScreenTape in the holder, with the QR code facing the reader. If the software warns you that the ScreenTape is expired, you can choose to ignore this warning and continue.
14. Place the PCR tube strips with your samples in the sample holder and remove the caps very carefully.
15. In the controller software, indicate the positions of your samples and specify sample IDs.
16. Press START and select a folder and filename for the data.
17. Wait while the samples are loaded and run.
18. TapeStation analysis software will launch automatically when the run is complete.

Equipment

• Heat block
• Incubator

Materials

• Competent cells (DH5𝞪/TOP10/BL21(DE3))
• SOC medium OR:
• LB medium
• Plasmid to insert

Procedure

1. Prepare a heat block at 42°C.
2. Take as many aliquot of competent cells (DH5𝞪/TOP10/BL21(DE3)) as needed from the -80°C freezer, and thaw on ice for 10-15 mins.
3. Add 15-20 µL of competent cells to each KLD sample.
4. Incubate on ice for 30 minutes.
5. Heat shock at 42°C for 45 s.
6. Put in 30s on ice.
7. Add 350 µL of SOC Medium.
8. Incubate at 37 °C for at least 1 hour. Shake vigorously (250 rpm) or rotate.
9. Plate the cells on a pre-warmed (at ~ 37 °C) LB agar plate with the desired antibiotic (Ampicillin in our case).
10. Incubate at 37 °C overnight.

Materials

• UTI software
• Wires
• Breadboard
• Pins

Procedure

1. Solder the pins to the board to connect the wires.
2. Connect the UTI to a laptop with a USB to USB A cable.
3. Select ‘Mode 1 (C23: 3 capacitors 0-2pF)’ within the program.
4. Connect the pins as described beneath:
1. Connect pin C to a reference capacitor of 1,8 pF.
2. Connect pin D to your electrode by making use of part of a cable (Ref. CACIDEP).
3. Connect pin A to the outputs of the capacitors from C and D. To reduce the influence of external factors, this connection can be made using coax cables.
4. Don’t connect pin B, E, and F.

The connection of the pins are visualized in Figure 1 .



5. Inside the software, change the reference capacitance to your reference capacitance (in our case 1.8 pF) and the average index to 50. The rest of the settings remain the initial conditions.

An overview of the settings and an image of the software is shown in Figure 2 .

Equipment

• Agilent 4294A Precision Impedance Analyzer (40 Hz - 110 MHz)

Procedure

1. Cut the electrodes between the two connection points in order to fit in the machine. See Figure 3 .
2. Place the electrode in the vector impedance analyzer.
3. Choose your desired start and end frequency for your sweep. In our case, this was 100 Hz to 1 MHz.
4. Set the marker at a frequency at which the capacitance was relatively stable but the resistance was still relatively high. In our case, this was 1 kHz.
5. Start measuring the ‘R-x’, the resistance and ‘C-p’, the parallel capacitance.
6. Measure the capacitance at your marker five times and note these values.
7. Take the average of these five values.