Introduction

This page contains a collection of wet lab protocols that our team used throughout the project. The purpose of this page is to present all the steps and reagents needed in the protocols in detail. To see the final results in detail, please go to the Results page. To learn more about our engineering approach, head over to the Engineering page.

Growth Media

In this protocol, you can find how to make LB-Miller medium and SOB/SOC medium. SOC medium can maximize the efficiency of competent cells during a transformation.

LB-Miller

• LB-Miller powder.
• Demineralised water.

1. Suspend 25 g of LB-Miller powder in 1 L demineralised water.
2. Autoclave.

SOB/SOC

• Tryptone.
• Yeast extract.
• NaCl.
• 1M KClact.
• Demineralized water.
• 1M MgCl2 solution.
• 1M MgSO4 solution.
• 20% glucose solution.

1. For 1000 mL final solution, mix the components listed in the table below.
2. Adjust to pH 7.0.
3. Fill up to 980 mL (to be completely precise), or to 1000 mL (if the previous option is not feasible).
4. Sterilize by autoclaving.
5. Before use (and after sterilization) add (sterile) components listed in the table below depending on whether you want to make SOB or SOC.

Basic components SOB/SOC medium:

SOB/SOC medium specific components:

Chemically competent cells (E. coli DH5-ɑ + TG1)

In this protocol, you will find how to make chemically competent cells. Make sure to work on ice from step 3 onwards!

• LB medium.
• 0.1 M CaCl2.
• Ice.
• ~86% glycerol.

1. Inoculate 50 mL LB medium with 0.5 mL overnight culture.
2. Grow the cells for approximately 2-3 hours at 37 ºC while shaking at 220 rpm until the OD600 is between 0.4-0.5. Take measurements each half hour.
3. Spin down cells at 4000 rpm for 10 minutes at 4 ºC. In all next steps: keep cells on ice.
4. Remove supernatant
5. Resuspend cell pellet in 25 mL ice-cold 0.1 M CaCl2.
6. Incubate cells on ice for 30 minutes.
7. Collect cell pellet by centrifugation at 4000 rpm for 10 minutes at 4 ºC.
8. Remove supernatant.
9. Carefully resuspend the cell pellet in 0.75 mL of 0.1 M CaCl2.
10. Add 129.31 µL ~86% glycerol.
11. Make aliquots of 100 µL and store cells at -80 ºC.

Electrocompetent cells (E. coli DH5-ɑ) and electroporation

In this protocol, you will find how to make electrocompetent cells.

• LB medium.
• 10% glycerol.
• Ice.
• Selective plates with the corresponding antibiotics.

1. Dilute 50 mL LB medium 1:50 with fresh overnight. of E. coli.
2. Follow the OD600.
3. At OD600 0.4-0.5, centrifuge at 8000 rpm, 4 ºC (from this moment: work as cold as possible).
4. Wash pellet successively with.
   • 40 mL ice-cold 10% glycerol.
   • 25 mL ice-cold 10% glycerol.
   • 10 mL ice-cold 10% glycerol.
5. Remove all supernatant and resuspend pellet in 100 µL ice-cold 10% glycerol.
6. Distribute aliquots of 50 µL in Eppendorf tubes, store at -80 ºC.

1. Cool the electroporation cuvette on ice.
2. Mix up to 5 µL DNA with 50 µL competent cells.
3. Add DNA/cell mix to the cuvette. Check that the cells are at the bottom of the cuvette and that it does not contain air bubbles.
4. Electroporate: 2.25 kV, 25 µF, 200 ohm.
5. Immediately afterwards: add 1 mL LB medium.
6. Incubate 1-1.5 hours at 37 ºC (preferably slow swirling).
7. Plate 100 µL dilutions on (selective) agar plates.
8. Incubate overnight at 37 ºC.

E. coli Transformation

In this protocol, you can find how to transform E. coli cells (how to insert the desired plasmid into E. coli) from competent E. coli cells aliquots.

• Ice.
• Prewarmed heat block at 42 ºC.
• Selective plates with the corresponding antibiotics.
• LB medium or SOC.
• Competent cells.
• Plasmid DNA.

1. Keep competent cells on ice when thawing.
2. Resuspend 5 µL of assembly product in 50 µL of cells in cold 1.5 mL Eppendorf tubes.
3. Leave cells on ice for 30 minutes.
4. Heat shock the cells for 30 seconds at 42 °C.
5. Leave cells on ice for 2 minutes.
6. Add 950 µL of SOC/LB.
7. Incubate at 37 °C in a shaker at ~220 rpm.
8. Spread 100-300 µL of cells onto (selective) agar plates.
9. Incubate the plates overnight at 37 °C.

Golden Gate Assembly Cloning using SapI

In this protocol, you can find how to perform a Golden Gate assembly protocol using SapI. This protocol is based on the New England BioLabs Golden Gate Assembly Protocol using SapI.

• T4 DNA Ligase.
• T4 DNA Ligase Buffer.
• SapI.
• Plasmid.
• DNA insert.
• MillQ.

1. Set up a 20 µL assembly reaction according to the table below.
2. Put the reaction mixture into a PCR machine for 1 hour at 37 °C.
3. Perform agarose gel electrophoresis or transformation to see whether the assembly was successful.

Components 20 µL Golden Gate assembly reaction:

Phusion Polymerase Chain Reaction (PCR)

In this protocol, you can find how to perform a Phusion polymerase chain reaction (PCR). This protocol is based on the New England BioLabs PCR Protocol for Phusion® High-Fidelity DNA Polymerase (M0530).

• 5x Phusion HF or GC buffer.
• dNTPs.
• Template DNA.
• Forward and reverse primer for your template DNA.
• Phusion DNA Polymerase.
• MilliQ.

1. Prepare the PCR reaction mixture based on the composition described in the table below. If you want to run multiple PCR reactions, it is convenient to make a Master Mix consisting of the 5x Phusion buffer, dNTPs, template DNA, Phusion DNA Polymerase, and MilliQ. You can make a primer mixture in MilliQ with 10 µM of forward primer and 10 µM of reverse primer and use 5 µL of that per 50 µL PCR reaction. Add the primer mixture to the Master Mix right before putting the samples in the PCR machine.
2. Mix the total PCR reaction mixture in the PCR tube gently by pipetting up and down.
3. Insert PCR tubes in the PCR machine using the settings depicted in the figure below. Perform 25-35 cycles of the denaturation, annealing, and extension steps. The annealing temperature was calculated using the New England BioLabs (NEB) Tm calculator.

Composition Phusion PCR reaction mixture for 50 µL PCR reaction:

Thermocycling conditions Phusion PCR:

Q5 Polymerase Chain Reaction (PCR)

In this protocol, you can find how to perform a Q5 polymerase chain reaction (PCR). This protocol is based on the New England BioLabs PCR Protocol for Q5® High-Fidelity 2X Master Mix.

• Q5 High-Fidelity 2X Master Mix.
• Forward and reverse primer for your template DNA.
• Template DNA.
• MilliQ.

1. Prepare the PCR reaction mixture based on the composition described in the table below. If you want to run multiple PCR reactions, it is convenient to make a Master Mix consisting of the Q5 High-Fidelity 2X Master Mix, template DNA, and MilliQ. You can make a primer mixture in MilliQ with 10 µM of forward primer and 10 µM of reverse primer and use 5 µL of that per 50 µL PCR reaction. Add the primer mixture to the Master Mix right before putting the samples in the PCR machine. For plasmid DNA, it is recommended to use 1 pg to 10 ng of the template DNA per 50 µL reaction.
2. Mix the total PCR reaction mixture in the PCR tube gently by pipetting up and down.
3. Insert PCR tubes in the PCR machine using the settings depicted in the figure below. Perform 25-35 cycles of the denaturation, annealing, and extension steps. The annealing temperature was calculated using the New England BioLabs (NEB) Tm calculator.

Composition Q5 PCR reaction mixture for 50 µL PCR reaction:

Colony PCR

In this protocol, you can find how to perform a colony polymerase chain reaction (PCR).

• Incubated plate with colonies.
• Prewarmed heat block at 95 ºC.
• Forward + reverse primers for DNA plasmid.
• Phire PCR master mix.
• MilliQ.

1. Choose between 8-16 colonies of the incubated plate of interest (+1 negative control), depending on how many plates you have.
2. Mark one 1.5 mL Eppendorf tube for each colony with ‘A’ (sterile) and another 1.5 mL Eppendorf for each colony ‘B’ (non-sterile).
3. Add 25 µL of sterile MilliQ to all A Eppendorfs.
4. Resuspend colonies in A Eppendorfs.
5. Transfer 12.5 µL of sample to B Eppendorfs.
6. Heat shock B Eppendorfs for 5 minutes at 95 °C in heat block.
7. Let B Eppendorfs rest on ice for 5 minutes.
8. Centrifuge B Eppendorfs at max speed for 2 minutes.
9. Add 3 µL of sample from B Eppendorfs to PCR tubes.
10. Make a master mix of 1 µL (10 µM) of forward primer, 1 µL (10 µM) of reverse primer, 5 µL of MilliQ, and 10 µL of 2x Phire green hot start II PCR mix for each reaction.
11. Run the Phire PCR program according to the settings in the table below. The annealing temperature can be calculated using the Tm calculator from the 2x Phire green hot start II PCR mix supplier.
12. Run samples on agarose gel electrophoresis to see if the desired DNA fragments are obtained.
13. If the desired DNA plasmid was found in a specific colony, use the corresponding Eppendorf A of that colony to prepare an overnight culture.

Components colony PCR 20 µL Reaction:

Thermocycling conditions Phire PCR:

Restriction/Diagnostic Digest

In this protocol, you can find how to perform a digest. Digests can be done to prepare DNA fragments for molecular cloning or to identify the identity of a plasmid.

• Miniprep kit.
• DNA.
• 10x rCutSmart Buffer.
• Appropriate restriction enzymes.
• MilliQ.

1. Miniprep overnight cultures. For the miniprep, we used the QIAprep(R) Spin Miniprep Kit from QIAGEN.
2. Measure plasmid concentration with NanoDrop.
3. Based on the plasmid concentration, you will add certain volumes of Plasmid, and master mix (made up of MilliQ, buffer, and enzymes). If you only use one restriction enzyme, you can add 0.25 µL (diagnostic) or 0.5 µL (for cloning). The total volume should be 25 µL or 50 µL.
4. Incubate at 37 °C for 1 hour.
5. Perform agarose gel electrophoresis to analyze the results.

Composition restriction digest reaction mixture for 25 µL and 50 µL reaction:

Ligation

In this protocol, you can find how to perform a ligation reaction.

• Insert DNA.
• Vector DNA.
• 10x T4 DNA Ligase Reaction Buffer.
• T4 DNA Ligase.
• MilliQ.

1. Use the New England BioLabs (NEB) Ligation Calculator to calculate how much vector (x) and insert (y) DNA is needed for an appropriate insert:vector ratio. The total volume of insert and vector DNA is a maximum of 17 µL.
2. Mix the components of the ligation reaction in a PCR tube in the order depicted in the table below (top to bottom). T4 DNA ligase should be added last!
3. Leave the ligation reaction for 1 hour at room temperature for the ligation reaction to occur.
4. Transform 5 µL of the reaction mixture into 50 µL competent cells to see whether the ligation reaction has worked.

Composition ligation reaction mixture for 20 µL ligation reaction:

Agarose Gel Electrophoresis

In this protocol, you can find how to perform agarose gel electrophoresis for the analysis of PCR results or digestion of plasmid results.

• Agarose.
• TAE buffer (1x).
• Gel electrophoresis set-up.
• SERVA DNA Stain G.
• To make 1% agarose gel:
• 500 mL TEA buffer.
• 5g agarose.
• Melt in the microwave until the mixture is clear.

1. Prepare the gel: Mix 40-50 mL of agarose gel mix (we used both 0.9% and 2%) with 0.8 µL dye (always use gloves). Pour the solution into the gel cast and insert combs. Wait for solidification (~20-40 minutes).
2. Mix the prepared sample with loading dye (Gel Loading Dye Purple (6x), NEB) (5:1 ratio, so for example 5 µL sample and 1 µL loading dye), and load a total volume of 5 µL or 10 µL on the gel. If the dye is already mixed in the sample (e.g. as for a colony PCR), pipette 5-10 µL of the samples directly into the well.
3. Add 5 µL of ladder (Quick-Load Purple 1 kb Plus DNA Ladder, NEB) to one of the wells (either first or last is convenient for analyzing the gel).
4. Run the gel at a constant 120 V for 30-40 minutes.
5. Analyze the gel using a GelDoc (Bio-Rad) imager.

Sequencing

In this protocol, you can find how to prepare samples for sequencing.

• Miniprep kit.
• 1.5 mL Eppendorf tubes.
• Overnight culture of bacteria containing plasmid.

1. Miniprep the overnight samples. For the miniprep, we used the QIAprep(R) Spin Miniprep Kit from QIAGEN.
2. Transfer 5 µL of the sample to a clean 1.5 mL Eppendorf and add 5 µL of 10 µM forward primer.
3. Transfer 5 µL of the sample to a (separate) clean 1.5 Eppendorf and add 5 µL of 10 µM reverse primer.
4. Label each of the Eppendorf tubes and send for sequencing.

Antibiotics concentrations

While working in the lab, we used different antibiotics for selection of our plasmids in E. coli. Here you can find an overview of the stock and working solutions.

• Stock solution: 100 mg/mL.
• Working solution: 100 µg/mL.
• Dilution factor: 1000.
• Results in: 1 µL per mL media.

• Stock solution: or 25 mg/mL.
• Working solution: 25 µg/mL.
• Dilution factor: 1000 .
• Results in: 1 µL per mL media.

• Stock solution: 50 mg/mL.
• Working solution: 50 µg/mL.
• Dilution factor:1000.
• Results in: 1 µL per mL media.

GFP Isolation

In this protocol, you can find how we isolated GFP to use for measurements with our electronic device.

• Overnight culture of bacteria containing GFP plasmid.
• LB medium.
• Y-PER.

1. Transfer 0.5 mL of an overnight culture containing the plasmid with GFP to 50 mL fresh LB medium.
2. Grow culture to OD = ~1.0.
3. Spin down cells.
4. Resuspend in 200 µL Y-PER.
5. Incubate cells for 20 minutes.
6. Spin down cells in a tabletop centrifuge for 20 minutes at full speed.
7. Collect supernatant and store in ~-20 °C freezer.