Team:TUDelft/Experiments

Sci-Phi 29

General Protocols

  • Annealing primers for G-block ﹀

    The primers to create G-blocks are provided with a concentration of 100 µM. The working stock for MoClo is 100 fmol/µL. Carry out a dilution to have a concentration of 0.1 µM. In order to anneal the primer for the G-block, follow the instructions below:

    1. Prepare the reaction mix:
      Component Volume (µL)
      Primer 1 9
      Primer 2 9
      Buffer* 10X 2
      Total 20
    2. Heat the reaction to 94 °C for 5 minutes.
    3. Let the reaction cool down to room temperature.

    The buffer 10X used for the annealing consists of:

    Component Final concentration (mmol/L)
    Tris 100 mmol/L
    NaCl 500 mmol/L
    EDTA 10 mmol/L

    The pH of the buffer should be between 7.5 - 8 and filter sterilize the buffer.

  • Antibiotic Stock Solution ﹀
    1. Weigh X gram of the antibiotic of interest.
    2. Dissolve the antibiotic of interest in Milli-Q water. The volume of Milli-Q water needed is determined by the desired antibiotic concentration.
    Note: Dissolve in 95% ethanol in case of chloramphenicol. We used the following antibiotic stock concentrations:     
    Antibiotic Stock Solution (mg/mL)
    Ampicillin 50
    Chloramphenicol 34
    Kanamycin 50
    Spectinomycin 50
  • Coupled IVTT and DNA replication ﹀
    1. For coupled In Vitro Transcription and Translation (IVTT), pipette the following volumes in a single PCR tube:
      2. Component Volume (µL)
      Solution I (Buffer) 7.5
      Solution II (Enzymes) 0.75
      Solution III (Ribosomes) 1.5
      Ammonium Sulfate (200mM) 1.5
      Purified p5 (10mg/mL) 1.125
      Purified p6 (7mg/mL) 0.45
      DNAP (2nM) X
      TP (2nM) Y
      Linear DNA (1nM) Z
      dNTPs 0.45
      RNAse inhibitor 0.375
      RNAse free Milli-Q 15 - X - Y - Z
      Total 15
    2. Incubate the nuclease-free PCR tubes at 30 °C for 8 hours

      3. To visualize the amplification of the linear DNA construct, you have to get rid of the RNA and DNA. This can be done by following the next steps:

    3. Treat with 0.5 µL RNAase A
    4. Treat with 1 µL RNAase I
    5. Incubate at 30 °C for 30 minutes
    6. Add 3 µL of EDTA stop solution
    7. Add 0.5 µL of Proteinase K
    8. Add 0.5 µL of Proteinase K
    9. Incubate at 30 °C for 30 minutes
    10. Incubate at 30 °C for 30 minutes
    11. Column purification
    12. Dry for 5 minutes with open lid
    13. Elute with 14 µL
    14. Load half (7 µL) with 3 µL loading dye on a 1% agarose gel
  • DNA Gel Electrophoresis ﹀

    The protocol for DNA gel electrophoresis includes mutagenic chemicals such as ethidium bromide (EtBR), SYBR Safe or another DNA staining. Carry out the protocol in an assigned area and wear gloves.


    Gel preparation:
    1. Prepare 0.8% (weight/volume) agarose in Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE) buffer.
    2. Heat the gel solution in the microwave to dissolve the agarose. Swirl the solution a few times while heating to ensure that the agarose powder is dissolved. The agarose is dissolved when the solution has bubbles and is clear.

    Casting of gel:
    1. Pick an appropriate gel stand and gel comb and place in the gel casting stand. Make sure to fasten the stand in place. Perform the next few steps quickly to prevent the gel from solidifying.
    2. Spread 15 µL (for a large stand) or 4-5 µl (for a small stand) of SYBR Safe or EtBr on the stand.
    3. Carefully pour the gel into the stand. Ensure that 3/4 of the teeth of the comb are submerged in the solution.
    4. Allow the gel to solidify. This will take approximately 30 minutes.

    Sample loading:
    1. After the gel has solidified, carefully remove the comb and transfer the gel to a gel box filled with TAE or TBE buffer.
    2. Cut a strip of parafilm. Pipette a drop of loading dye onto the parafilm, then pipette the sample on top of the loading dye. The ratio should be 1:5:dye:sample (e.g. 1 µL of dye and 5 µL of sample). Mix the solutions by pipetting up and down a few times. Repeat this step for each sample.
    3. Load the samples carefully in the slots. Make sure not to poke the gel.
    4. Load 3 µL of DNA ladder in a separate slot.
    5. Set the voltage to 100 volt.
    6. Run the gel until the DNA has moved across 2/3 of the gel. This will take approximately 45 minutes.
    7. Visualize the gel using a gel documentation system (gel doc).
  • DNA Gel Purification ﹀

    Protocol for DNA gel purification using Promega Wizard® SV Gel and PCR Clean-Up System:


    Dissolving the gel slice:

    1. Following electrophoresis, excise a DNA band from the gel and place this gel slice in a 1.5 mL microcentrifuge tube.
    2. Add 10 µL of Membrane Binding Solution per 10 mg of gel slice. Vortex and incubate at 50–65 °C until the gel slice is completely dissolved.

    Binding of DNA:

    1. Insert a Wizard® SV Minicolumn into a Collection Tube.
    2. Transfer the dissolved gel mixture to the Minicolumn assembly. Incubate at room temperature for 1 minute.
    3. Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert the Minicolumn into the Collection Tube.

    Washing:

    1. Add 700 µL of Membrane Wash Solution. Membrane Wash Solution should be prepared beforehand with 15 mL of 95% ethanol added per 3 mL of concentrated Membrane Wash Solution. Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert the Minicolumn into the Collection Tube.
    2. Repeat step 1 with 500 µL of Membrane Wash Solution. Centrifuge at 16,000 × g for 5 minutes.
    3. Empty the Collection Tube and centrifuge the column assembly again for 1 minute at 16,000 x g with the microcentrifuge lid open or off to allow evaporation of any residual ethanol.

    Elution:

    1. Carefully transfer the Minicolumn to a clean 1.5 mL microcentrifuge tube.
    2. Add 50 µL of nuclease-free water to the Minicolumn. Incubate at room temperature for 1 minute. Then centrifuge at 16,000 × g for 1 minute.
    3. Discard the Minicolumn and store the microcentrifuge tube holding the DNA at 4 °C or –20 °C.
  • Expressing phi29 proteins in PUREfrex system ﹀
    1. Thaw DNA and Purefrex 2.0 on ice
    2. Pipette the following volumes in a single PCR tube
      Component Volume (µL)
      Solution I (Buffer) 5
      Solution II (Enzymes) 0.5
      Solution III (Ribosomes) 1
      DNA (5nM) X
      Green Lys 0.5
      RNAse free Milli-Q 3 - X
      Total 10
    3. Incubate the nuclease-free PCR tubes at 37 °C for 3 hours
    4. Treat the expressing sample with RNAase (RNaseA Solution, Promega), place the sample at 37 °C for 30 min
    5. Denaturate the protein in the sample for 10 minutes at 90 °C in 2x SDS loading buffer with 10 mM dithiotreitol (DTT)
    6. Load 8 µL on a SDS PAGE
    7. SDS PAGE visualization: To visualize the fluorescently labeled phi29 proteins a fluorescence gel imager (Typhoon, Amersham Biosciences) was used with a a 488-nm laser and a band pass emission filter of 520 nm.
  • FACS ﹀

    1. Pick a single colony of a clone and inoculate in 5 mL LB with respective antibiotic in a 50 mL falcon tube.
    2. Incubate the culture overnight (for E. coli at 37 ° celsius overnight and 30 ° celsius for P. putida) at 250 rpm.
    3. Dilute the overnight culture 1:100 in 2 mL eppendorf tubes.
    4. Incubate the culture at 37 or 30 ° celsius and 250 rpm for 2 hours.
    5. After 2 hours, dilute the culture 1:100 in 5 mL medium in 50 mL falcon tubes.
    6. Incubate the culture at 37 ° celsius and 250 rpm for 4 hours.
    7. After 4 hours of incubation, keep the cells in the fridge for an hour.
    8. Dilute the sample 1:10 in PBS. The total sample volume can be 20 µl.
    9. Analyze your sample on FCSalyzer v.0.9.18-alpha with settings appropriate for bacterial cultures.
  • Gibson Assembly ﹀

    The Gibson Assembly is based on the protocol provided by New England Biolabs.


    1. Thaw 10 µL of 2X Gibson Assembly Master Mix on ice.
    2. Add DNA vector and insert. The recommended ratio of mol vector to mol insert is 1:3. The volume of vector and insert together should not exceed 10 µL.
      Component Volume (µL)
      Gibson Assembly Master Mix 2X (NEB) 10
      Vector X
      Insert Y
      Milli-Q 10 - X - Y
      Total 20
    3. Incubate the assembly reaction at 50 °C for 1 hour.
  • GFP Measurement in Gel Doc ﹀
    1. Measure the OD of the cell culture and dilute the cell cultures to an OD of 1.
    2. Centrifuge the cell for 1 minute at max rpm.
    3. Discard the supernatant.
    4. Measure the fluorescence with the GFP fluorescence measurement setting installed in the gel doc.
  • IPTG Induction ﹀
    Day 1:
    1. Inoculate 10 mL of liquid LB medium containing the right selection marker with the cells of interest from the glycerol stock.
    2. Incubate the liquid media at 37 ° C and 250 rpm overnight
    Day 2:
    1. Inoculate 1 mL of fresh LB media into an Eppendorf tube containing the right selection marker with 1 µL of the overnight grown culture.
    2. Incubate the cells at 37 ° C and 250 rpm till the OD of the cell culture reaches 0.4
    3. Add 2 µL of 1 mM IPTG to the cell culture and incubate the cells overnight at 20 ° C and 250 rpm.
  • IPTG Stock ﹀
    1. Mix 238 mg of IPTG with 900 µl of ddH2O (we used Milli-Q).
    2. Add ddH2O until final volume is 1 mL.
    3. Filter sterilize with a 0,22 micro syringe filter (the ones that we have are 25 and that is fine) Store at -20 °C.
  • Ligation ﹀
    1. Thaw the ligase buffer on ice.
    2. Add the following components together:
      Component Volume (µL)
      10X ligase buffer 2
      T4 DNA ligase 1
      DNA vector X (~100 ng)
      DNA fragment Y*
      Milli-Q 17 - X - Y
      Total 20

      3. *The recommended vector to insert ratio is 1:3.

    3. Incubate at 4 °C for at least 1 hour. Optimally, incubate overnight at 4 °C.
  • Mass Spectrometry Preparation ﹀

    To prepare PUREfrex reaction for Mass Spectrometry follow these steps:

    1. Add the following to a tube
      2. Component Volume (µL)
      Cell free expression sample 5
      100 mM Tris pH 8 6.25
      250 mM DTT 0.4
      20 mM CaCl2 1.25
      Milli-Q 9.775
      PURE buffer 0
      Sample after using following Expressing phi29 proteins inPUREfrex system 2
    2. Incubate for 10 minutes on 90 °C
    3. Add 1.5 µl 250 mM IAM to the tube
    4. Incubate for 30 minutes in a dark space
    5. Add 0.4 µl 250 mM DTT and 0.625 Trypsin (1 mg/ml) to the tube
    6. Incubate overnight at 37 ° C
    7. Add 2.52 10% TFA (or formic acid) to the tube
    8. Centrigue for 10 minutes at max rpm
    9. Transfer to glass vial with insert
    10. Sample is ready to be put in Mass Spectrometer
  • Modular Cloning (MoClo) ﹀

    Modular cloning (MoClo) based on the Golden Gate cloning protocol.


    Pipette the following volumes in a single PCR tube for MoClo level 0:

    Component Volume (µL)
    DNA insert (40 fmol) 1
    Vector (40 fmol) 1
    T4 DNA ligase buffer (NEB) 1
    T4 DNA ligase (NEB) 0.5
    BpiI 0.5
    Nuclease-free water 6
    Total 10

    Pipette the following volumes in a single PCR tube for MoClo level 1 and 2:

    Component Volume (µL)
    Each DNA insert (40 fmol) 1
    Vector (40 fmol) 1
    T4 DNA ligase buffer (NEB) 1
    T4 DNA ligase (NEB) 0.5
    Level 1: BsaI V2 (NEB)
    Level 2: BpiI     		0.5
    Nuclease-free water Amount needed for a total volume of 10 µL
    Total 10

    Place the PCR tube in a thermocycler with the following protocol for level 0 assembly:

    Step Temperature ( °C) Time
    1 37 2 minutes
    2 16 5 minutes
    Repeat step 1 and 2 50 times.
    3 50 10 minutes
    4 80 10 minutes

    Place the PCR tube in a thermocycler with the following protocol for level 1 assembly:

    Step Temperature ( °C) Time
    1 37 20 minutes
    2 16 3 minutes
    3 37 1.5 minutes
    Repeat step 1 and 2 15 times.
    4 37 20 minutes
    5 50 5 minutes
    5 50 5 minutes

    Place the PCR tube in a thermocycler with the following protocol for level 2 assembly:

    Step Temperature ( °C) Time
    1 37 5 minutes
    2 16 10 minutes
    Repeat step 1 and 2 30 times.
    4 37 1 hour
    5 60 5 minutes
    5 8 ∞

  • Nanodrop DNA Quantification ﹀
    1. Use a Nanodrop spectrometer to quantify the DNA amount and purity in a DNA purification or extraction sample.
    2. Adjust the settings to measure double-stranded DNA (dsDNA) at 260 nm and impurities at 230 nm and 280 nm.

    Note: DNA should have a 260/280 between 1.8 and 2.0. If the value is below 1.8 it means the sample is contaminated with extraction agents, such as phenol or ethanol.

  • Polymerase Chain Reaction (PCR) ﹀
    • Colony PCR (GoTaq) ﹀
      1. Add the following components for a colony PCR in a PCR tube:

        2. Component Volume (µL) Final concentration
        GoTaq 5X buffer 10 1x
        10 mM dNTPs 1 200 µM
        Forward primer 1 200 nM
        Reverse primer 1 200 nM
        Colony* NA NA
        GoTaq Polymerase (5 U/µL) 0.2 20 U/mL
        Nuclease-free water 36.8 NA
        Total 50

        *Pick a colony under aseptic conditions and resuspend it in Milli-Q water.


      2. Place the PCR tube in a thermocycler with the following protocol:
        3. Step Temperature ( °C) Time
        Initial denaturation 98 150 sec
        Denaturation 94 60 sec
        Annealing Tannealing* 60 sec
        Extension 72 1 min/kb DNA
        Repeat the steps from 'Denaturation' to 'Extension' 29 times.
        Final extension 72 5 minutes
        Hold 4 ∞

        *The annealing temperature is dependent on the melting temperature (Tm) of the primers. Tannealing = Tm - 5 °C.

      3. Check the PCR product on an agarose gel (see DNA electrophoresis protocol).
    • Phusion PCR ﹀
      1. Add the following components for a Phusion PCR in a PCR tube:

        2. Component Volume (µL) Final concentration
        5X Phusion HF buffer 10 1x
        10 mM dNTPs 1 200 µM
        Forward primer 1 200 nM
        Reverse primer 1 200 nM
        Template DNA (10 ng) X Varies
        Phusion polymerase 0.5 20 U/mL
        Nuclease-free water 36.5 - X NA
        Total 50

      2. Place the PCR tube in a thermocycler with the following protocol:
        3. Step Temperature ( °C) Time
        Initial denaturation 98 30 sec
        Denaturation 98 10 sec
        Annealing Tannealing* 15 sec
        Extension 72 30 sec/kb DNA
        Repeat the steps from 'Denaturation' to 'Extension' 29 times.
        Final extension 72 5 minutes
        Hold 20 ∞

        *The annealing temperature is dependent on the melting temperature (Tm) of the primers. Tannealing = Tm - 5 °C.

      3. Check the PCR product on an agarose gel (see DNA electrophoresis protocol).
    • KOD Xtreme PCR ﹀
      1. Add the following components for a KOD Xtreme PCR in a PCR tube:

        2. Component Volume (µL) Final concentration
        2x Novagen KOD Xtreme buffer 2 1x
        10 mM dNTPs 2 400 µM
        Forward primer 1.5 300 nM
        Reverse primer 1.5 300 nM
        Template DNA (100 ng) X Varies
        Novagen KOD polymerase 1
        Nucleas-free water 36.5 - X NA
        Total 50

      2. Place the PCR tube in a thermocycler with the following protocol:
        3. Step Temperature ( °C) Time
        Initial denaturation 94 2 minutes
        Denaturation 98 10 sec
        Annealing 68 30 sec
        Extension 68 4 minutes
        Repeat the steps from 'Denaturation' to 'Extension' 29 times.
        Final extension 68 7 minutes
        Hold 12 ∞
      3. Check the PCR product on an agarose gel (see DNA electrophoresis protocol).
  • PCR Clean-up ﹀

    Protocol for PCR clean-up using Promega Wizard® SV Gel and PCR Clean-Up System:


    Processing PCR amplifications:

    1. Add an equal volume of Membrane Binding Solution to the PCR amplification.

    Binding of DNA:

    1. Insert an SV Minicolumn into a Collection Tube.
    2. Transfer the prepared PCR product to the Minicolumn assembly. Incubate at room temperature for 1 minute.
    3. Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert the Minicolumn into the Collection Tube.

    Washing:

    1. Add 700 µL of Membrane Wash Solution. Membrane Wash Solution should be prepared beforehand with 15 mL of 95% ethanol added per 3 mL of concentrated Membrane Wash Solution. Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert the Minicolumn into the Collection Tube.
    2. Repeat step 1 with 500 µL of Membrane Wash Solution. Centrifuge at 16,000 × g for 5 minutes.
    3. Empty the Collection Tube and centrifuge the column assembly again for 1 minute at 16,000 x g with the microcentrifuge lid open or off to allow evaporation of any residual ethanol.

    Elution:

    1. Carefully transfer the Minicolumn to a clean 1.5 mL microcentrifuge tube.
    2. Add 50 µL of nuclease-free water to the Minicolumn. Incubate at room temperature for 1 minute. Then centrifuge at 16,000 × g for 1 minute.
    3. Discard the Minicolumn and store the microcentrifuge tube holding the DNA at 4 °C or –20 °C.
  • Plasmid Isolation ﹀

    Protocol for plasmid isolation using Promega PureYield™ Plasmid MiniPrep System:

    Prepare lysate:

    1. Pipette 1.5 mL of the bacterial culture in an Eppendorf tube and centrifuge for 30 seconds at maximum speed in a microcentrifuge.
    2. Discard the supernatant.
    3. Repeat steps 1 and 2 until the entire inoculum volume has been spun down.
    4. Add 600 µL of Milli-Q water to the cell pellet and resuspend completely.
    5. Add 100 µL of Cell Lysis Buffer and mix by inverting the tube 6 times. The solution should change from opaque to clear blue, indicating complete lysis.
      6. Notes:
      1. If you have more sample volume, split it. This will help complete this step quickly.
      2. Proceed to step 6 within 2 minutes. Excessive lysis can result in denatured plasmid DNA. If processing a large number of samples, process samples in groups of ten or less. Continue with the next set of ten samples after the first set has been neutralized and mixed thoroughly.
    6. Add 350 µL of cold (4–8 °C) Neutralization Solution and mix thoroughly by inverting the tube. The sample will turn yellow when neutralization is complete, and a yellow precipitate will form. Invert the sample an additional 3 times to ensure complete neutralization.
    7. Centrifuge at maximum speed in a microcentrifuge for 3 minutes.
    8. Transfer the supernatant (~900 µL) to a PureYield™ Minicolumn. Do not disturb the cell debris pellet. For maximum yield, transfer the supernatant with a pipette.
    9. Place the minicolumn into a PureYield™ Collection Tube and centrifuge at maximum speed in a microcentrifuge for 15 or 30 seconds.
    10. Discard the flowthrough and place the minicolumn into the same PureYield™ Collection Tube.

    Wash:

    1. Add 200 µL of Endotoxin Removal Wash to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 15 seconds. It is not necessary to empty the PureYield™ Collection Tube.
    2. Add 400 µL of Column Wash Solution to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 30 seconds.

    Elute:

    1. Transfer the minicolumn to a clean 1.5 mL microcentrifuge tube, then add 30 µL of nuclease-free water at neutral pH directly to the minicolumn assembly. Let stand for 1 minute at room temperature.
      2. Notes:
      1. Elution buffer can also be used, but it can interfere with future cloning steps.
      2. For large plasmids (>10 kb), warm the Elution Buffer to 50ºC prior to elution, and increase elution volume to 50 µL. Also incubate the column at room temperature (22–25 °C) for 5–10 minutes before proceeding to the next step.
    2. Centrifuge at maximum speed in a microcentrifuge for 15 seconds to elute the plasmid DNA. Cap the microcentrifuge tube and store eluted plasmid DNA at –20 °C.
    3. Follow the Nanodrop DNA quantification protocol to determine the purity and yield of your plasmid isolation.
  • Restriction Digestion ﹀
    1. Choose the desired enzyme and its required buffer. The CutSmart buffer 10X is used for most NEB restriction enzymes.
    2. Add the following components together:
      Component Volume (µL)
      10X CutSmart buffer (NEB) 2
      Fragment (~1-2 μg) X
      Restriction enzyme(s) 1 of each enzyme
      Milli-Q 18 - X - added restriction enzyme
    3. Incubate for 4 hours at 37 °C.
    4. Inactivate the restriction enzyme by heating to 65 °C for 10 minutes.
  • SDS PAGE ﹀

    SDS PAGE preparation: An SDS PAGE gel consist of a stacking gel solution and a resolving gel solution. In this protocol we use a 4% Stack gel and a 18% resolving gel (this is for protein samples between 10 kDa and 100 kDa).


    1. Prepare the following gels:
      Component 3x stacking gel solution 4% 3x resolving gel solution 18%
      1.5M Tris-HCl (pH 8.8) - 3.75 mL
      0.5M Tris-HCl (pH 8.8) 1.25 mL -
      20% SDS w/v 0.075 mL 0.025 mL
      AA/BAA 37.5%/0.8% 7.2 mL 0.53 mL
      10% APS 0.075 mL 0.025 mL
      TEMED 0.01 mL 0.005 mL
      Milli-Q 3.9 mL 3.2 mL
      This is enough to make 3 gels
    2. Assemble the moulds and pour the resolving gel in the mould
    3. When the gel is solidified, the stacking gel can be poured on top and the gel comb can be placed
    4. Let it solidify, then remove the comb. The gel is now ready to use, but can also be stored at 4 °C in hoisted paper towels.

    Running SDS PAGE gel: Run the gel at 100 Volt for 15 minutes and after that run it at 180 Volt for 1 hour


Strain Specific Protocols

  • Bacillus subtilis ﹀
    • Electrocompetent Cells Preparation ﹀
      1. Dilute an overnight culture of Bacillus subtilis 16-fold in growth medium and grow at 37 °C to an OD 600 of 0.85-0.95.
      2. Cool the cells on ice water for 10 minutes and harvest by centrifugation at 4 °C and 5000 x g for 5 minutes.
      3. Wash cells four times in an ice-cold electroporation medium.
      4. Suspend the cells in 1/40 of the culture volume of the electroporation solution with a cell concentration of 1.0-1.3 x 1010 cfu/mL.
      5. The competent cells can be stored at –80 °C until use with some decrease in transformation efficiency.
    • Transformation by Electroporation ﹀
      1. Add 1 µL (50 ng/µL) of plasmid DNA to 60 µL of electrocompetent cells. Homogenize by gently pipetting up and down several times.
      2. Transfer mixture into a pre-chilled cuvette. Incubate for 1.0 - 1.5 minutes.
      3. Wipe moisture from the cuvette and insert the cuvette into an electroporation device.
      4. Electroporation settings:
        5. Mode Prokaryotes "O"
        Voltage 2,100 V
        Time constant (τ) 5 ms
        No. of pulses (n) 1
      5. Immediately add 1 mL outgrowth medium and incubate for 3 hours at 37 °C.
      6. Plate onto selective LB agar plates and incubate overnight at 37 °C.
  • Escherichia coli ﹀
    • Chemically Competent Cells Preparation ﹀

      The protocol for chemically competent cells has a duration of three days, starting from the preparation of the reagents. Throughout the protocol, it is mandatory to work under aseptic conditions to prevent contamination risks as much as possible.

        Day 1 (preparation):
      1. Prepare the necessary reagents and sterilize the reagents and centrifuge bottles.
        • 1 L Luria-Bertani (LB) medium
        • 10 mL Luria-Bertani (LB) medium
        • 400 mL 100mM CaCl2
        • 20 mL 100 mM CaCl2 + 15% glycerol
      2. Store the CaCl2 and the CaCl2 + 15% glycerol stock solution at 4°C for at least 24 hours.
      3. Take the strain of interest from the -80°C freezer and store it on ice.
      4. Streak out the desired E. coli strain on an LB plate with antibiotic selection marker and incubate at 37°C overnight.

        5. Day 2 (preparation):
      5. Inoculate one single colony from the overnight grown plate in 10 mL LB medium with antibiotic selection marker overnight at 37°C while shaking at 200 rpm.

        6. Day 3:
      6. Inoculate 10 mL from the overnight grown liquid cell culture in 1 L of LB medium with antibiotic selection marker. Grow the inoculated medium at 37°C while shaking at 200 rpm until the OD 600 is between 0.4 and 0.6. The OD is measured for the first time after 1.5 hours after inoculation. (Important: the OD should not rise above 0.6 in order to obtain good competent cells)
        7. Notes:
        1. From this point on be very careful with your cells. The cells are very fragile.
        2. Everything that touches the cells should not be warmer than 4°C.
        3. Always keep the cells on ice. Resuspension steps also need to be performed on ice.
        4. Work clean and fast, the cells heat up very quickly which influences the quality of the competent cells a lot.

      7. Divide the 1 L of LB medium with cells into centrifuge cylinders. Weigh them to ensure equal weight distribution during centrifugation. Prepare a counterweight if necessary.
      8. Harvest the cells by centrifugation for 5 minutes at 4000 rpm at 4°C. Carefully resuspend the pellets in 200 mL ice-cold CaCl2 using an electronic pipet.
      9. Incubate the resuspended cells on ice for 20 minutes.
      10. Harvest the resuspended cells by centrifugation for 5 minutes at 3000 rpm at 4°C. Gently resuspend the new pellet in 100 mL ice-cold CaCl2 using an electronic pipet.
      11. Incubate the resuspended cells on ice for 1 hour. In the meantime, mark approximately 75 1.5 mL Eppendorf tubes and store them on ice.
      12. Harvest the resuspended cells by centrifugation for 5 minutes at 3000 rpm at 4°C. Gently resuspend the new pellet in 10 mL ice-cold CaCl2 + 15% glycerol using an electronic pipet.
      13. Aliquot 50 μL into sterile 1.5 mL microfuge tubes that you marked previously and snap freeze with liquid nitrogen. Since the cells are very fragile, cut the pipet tips in order to reduce stress on the cells.
      14. Store the frozen cells in the -80°C freezer.
      15. Immediately perform a heat shock transformation to determine the transformation efficiency.
    • Glycerol Stock Preparation﹀
      1. Incubate ~50 µL of cells in 5 mL LB medium with selection marker overnight.
      2. Pipette 1.5 mL in an Eppendorf tube and spin down the bacterial culture.
      3. Discard the supernatant.
      4. Repeat step 2 and 3.
      5. Resuspend the pellet in 0.9 mL liquid bacterial culture.
      6. Add the liquid bacterial culture from the Eppendorf tube to a cryotube.
      7. Add 0.9 mL 50% glycerol to the cryotube and mix well.
      8. Store at -80°C.
    • Luria-Bertani (LB) Agar Preparation ﹀
      1. Dissolve 14.24 g of LB agar in 400 mL of Milli-Q water.
      2. Autoclave the LB agar solution.
    • Luria-Bertani (LB) Medium Preparation ﹀
      1. Dissolve 20 g/L Luria-Bertani (LB) powder in water as described in the instructions provided by the manufacturer.
      2. Autoclave the medium at 121 °C to heat sterilize.
      3. Store the sterilised LB medium at room temperature.
    • Heat Shock Transformation ﹀
      1. Thaw 50 µL of chemically competent cells on ice for 10 minutes.
      2. Add 0.1-10 ng of plasmid DNA or 20-50 ng of ligate to the competent cells. Incubate on ice for 30 minutes.
      3. Heat shock for 45 seconds at 42 °C.
      4. Incubate on ice for 2 minutes.
      5. Add 1 mL of LB medium and mix gently.
      6. Incubate for 1 hour at 37 °C.
      7. Prepare agar plate with the right corresponding antibiotic marker. Use 25 mL agar with 200 µL of antibiotic stock solution.
      8. Plate 100 µL of cell suspension on the agar plate and grow it overnight at 37 °C.
  • Pseudomonas putida ﹀
    • Electrocompetent Cells Preparation ﹀
      1. Inoculate 50 mL LB medium with 7 mL of a fresh overnight culture of Pseudomonas putida. Grow cells at 30 °C to an OD 600 of 0.8.
      2. Harvest by centrifugation at 4000 rpm for 5 minutes at 4 °C.
      3. Wash twice with 50 mL of ice-cold glycerol. Centrifuge at 3000 rpm for 5 minutes at 4 °C.
      4. Resuspend cells in 0.8 mL of ice-cold glycerol. Keep on ice.
    • Transformation by Electroporation ﹀
      1. Add 4 µL of plasmid DNA (1 µg) to 40 µL of electrocompetent cells. Homogenize by gently pipetting up and down several times. Transfer mixture into a prechilled cuvette.
      2. Wipe moisture from the cuvette and insert the cuvette into an electroporation device.
      3. Electroporation settings:
        4. Mode Prokaryotes "O"
        Voltage 2,400 V
        Time constant (τ) 5 ms
      4. Immediately add 1 mL of LB medium. Incubate for 2 hours at 30 °C.
      5. Plate cells on selective plates.
  • Vibrio natriegens ﹀
    • Electrocompetent Cells Preparation ﹀
      1. Grow a 10 mL culture of V. natriegens cells in BHIv2 (BHI with v2 salt: 204 mM NaCl, 4.2 mM KCl, and 23.14 mM MgCl2) overnight.
      2. Inoculate 100 mL of BHIv2 with 1 mL of overnight culture.
      3. Place in an orbital shaker at 37 °C at 200 rpm until OD 600 reaches 0.5.
      4. Separate culture into two 50 mL falcon tubes and place on ice for 15 minutes.
      5. Pellet cells at 6500 rpm for 20 minutes at 4 °C.
      6. Resuspend the cells in each tube in 5 mL (10 mL total) of electroporation buffer (680 mM sucrose, 7 mM K2HPO4, pH 7).
      7. Fill each tube to ~35mL of electroporation buffer and invert gently to mix.
      8. Centrifuge cells at 6500 rpm for 15 minutes at 4 °C.
      9. Decant supernatant with a pipette.
      10. Wash the cells with electroporation buffer as described above a total of three times.
      11. After the final wash, resuspend cells in electroporation buffer as to have an OD 600 of 16.
      12. Make aliquots of 100 µL of the cells and store at -80 °C.
    • Transformation by Electroporation ﹀

        Transformation in Vibrio natriegens is based on the following protocol Vmax™ Express Electrocompetent Cells User Guide, 7 Synthetic Genomics Inc.

      1. For each transformation reaction,
        • Thaw one vial of Electrocompetent cells on ice for 5 minutes.
        • Warm one vial of BHIv2 (BHI with v2 salt: 204 mM NaCl, 4.2 mM KCl, and 23.14 mM MgCl2) to room temperature (20 °C to 24 °C).
        • Pre-chill a 0.1 cm electroporation cuvette on ice.
      2. As soon as the electrocompetent cells are thawed, add 10 ng – 100 ng of plasmid DNA (in TE buffer or water only) to the competent cells on ice. Briefly mix by flicking tubes. Note: Add no more than 2 μL of plasmid DNA to the competent cells. Concentrate the plasmid DNA if necessary.
      3. Incubate the DNA and cell mixture on ice for 3–5 minutes.
      4. During incubation, set the following electroporation parameters: 900 V, 25 uF, 200 Ohms, 0.1 cm cuvette. If further optimization is desired, voltage changes in the range of +/- 200V in 50 V increments can be used. Note: These are the optimized electroporation settings for the Gene Pulser® XcellTM Microbial System using a cuvette with a 0.1 cm gap. If other instruments and/or cuvettes are used, you may need to optimize the conditions for best results.
      5. Transfer cells to a chilled cuvette using a pipette. Verify that the cells completely occupy the bottom of the cuvette and that no air remains between walls of the cuvette and the cells.
      6. Transfer the cuvette to the shock pod and perform electroporation.
        • Note: Time constants typically range from 3.3–3.7 ms.
      7. Add 500 uL of BHIv2 recovery media to the cuvette immediately after pulse.
      8. Transfor the BHIv2 media and cell mixture to an Eppendorf tube and place in an orbital shaker (250 rpm) at 30 °C for 2 hours at to allow recovery of cells and expression of antibiotic resistance marker.
        Note: Recovery can be performed at 37 °C unless kanamycin is used for selection.
      9. During cell recovery, pre-warm LB-Miller agar plates prepared with antibiotics at the appropriate incubation temperature.

      Plating notes
      • We recommend incubating plates at 25 °C to 30 °C overnight.
      • For plates containing kanamycin, plates must be incubated at 25 °C to 30 °C.
      • Colonies typically appear after 12–16 hours at 25 °C to 30 °C and after ~8 hours at 37 °C.
      • Store agar plates with established VmaxTM Express at room temperature for up to 72 hours. Due to contact inhibition single colonies will remain well-isolated.
      • Do not store VmaxTM agar plates at 2–8 °C as this can adversely impact propagation and growth.
      • For long-term storage, we recommend preparing glycerol stocks with 25% glycerol and storing at −80 °C.


      Antibiotic Solid media Liquid culture
      Ampicillin 2-50 µg/mL 2-25 µg/mL
      Kanamycin 100 µg/mL 200 µg/mL
      Tetracycline 2.5 µg/mL
      Chloramphenicol 5–12.5 μg/mL 12.5–25 μg/mL