Consumables

• Kanamycin
• Ampicillin
• Tetracycline
• Sterile milliQ
• 70% ethanol
• 10 mL or 20 mL syringe
• 50 mL falcon tubes
• Syringe filter
• Sterile 1.5 mL Eppendorf tubes

Equipment

• Vortex

Protocol

• Weigh needed amount of antibiotic and put it in a falcon tube.
• Add 20 mL sterile milliQ or 70% ethanol to the falcon tube with antibiotic.
• Vortex until dissolved.
• Filter antibiotic stock solution through 0.2 µm filter.
• Aliquot in 1.5 mL Eppendorf tubes, label and store at -20 °C.

Based on: Peyraud R, Kiefer P, Christen P, Massou S, Portais JC, Vorholt JA. Demonstration of the ethylmalonyl-CoA pathway by using 13C metabolomics. Proc Natl Acad Sci. 2009;106(12):4846-4851. doi:10.1073/pnas.0810932106

Consumables

• NH₄Cl
• MgSO₄ • 7 H₂O
• K₂HPO₄
• NaH₂PO₄ • 2 H₂O
• Agarose
• Na₂EDTA • 2 H₂O
• FeSO₄ • 7 H₂O
• NaOH and HCl for pH adjustment
• ZnSO₄ • 7 H₂O
• CoCl₂ • 6 H₂O
• MnCl₂
• H₃BO₃
• Na₂MoO₄ • 2 H₂O
• CuSO₄ • 5 H₂O
• CaCl₂ • 2 H₂O
• Sterile methanol
• Millipore water and/or milliQ water

Equipment

• Autoclave
• Filter
• Vacuum pomp
• Sterile 1 L flasks
• Pipette tips, pipettes, pipetboy
• Downflow cabinet

Protocol

Mineral salts stock solution

• Prepare the mineral salts stock solution. Dissolve the salts in MilliQ water. Sterilize by autoclaving.

Buffer pH 7.1

• Prepare the mineral salts stock solution. Dissolve the salts in MilliQ water. Sterilize by autoclaving.

3% agarose

• For making plates with minimal medium, prepare 3% agarose. Dissolve agarose in MilliQ water and sterilize by autoclaving.

Iron solution (1000x)

• Prepare the iron stock solution. Add EDTA and FeSO4 to MilliQ and adjust pH to 4.

Trace element solution (1000x)

• Prepare the trace element stock solution. Add all trace elements and adjust to pH 1-2 until all salts are dissolved.

Medium preparation

• Work sterile in the downflow cabinet. Add the following ingredients in the respective order to a sterile 1 L flask to prepare 1 L mineral medium.

• Run the solution through a 2 µm filter using a vacuum pomp into a new 1 L flask. Do not let the filter run dry.

Consumables

• Petridish
• Antibiotic stock 1000x working concentration
• Autoclaved medium (LB agar, M. extorquens Minimal medium, etc)

Equipment

• Sterile environment (laminar flow cabinet with HEPA filter, bunsen burner, etc)

Protocol

For 10 plates

1. Microwave 250 mL medium (LB agar).
2. Let cool and add 250 µL 1000x antibiotic stock.
3. Pour plates in sterile environment (10 plates per 250 mL).
4. Label with red marker for tetracycline, blue marker for ampicillin or green marker for kanamycin.
5. Let cool and store in fridge in plastic, labelled with medium (LB agar) + individual who poured it + [antibiotic] + date.

Based on: Addgene, Protocol - How to run an Agarose gel. https://www.addgene.org/protocols/gel-electrophoresis/.

Consumables

• 1x TAE buffer
• Agarose (analytical grade)
• Ethidium bromide (stock concentration of 10 mg/mL)

Equipment

• Casting tray
• Well combs
• Voltage source
• Gel box
• UV light source/Gel doc gel photography machine
• Microwave

Protocol

Making a gel

1. Measure 2.5 g of agarose.
2. Mix agarose powder with 250 mL 1xTAE in a microwavable flask.
3. Microwave until the agarose is fully dissolved. Do not overboil, as some of the buffer will evaporate and thus alter the final percentage of agarose in the gel. It is best to microwave in pulses and swirl the flask occasionally as the solution heats up.



CAUTION: Hot! Be careful stirring, eruptive boiling can occur.

4. Let agarose solution cool down to about 50 °C (about when you can comfortably keep your hand on the flask), about 5 mins.
5. Pour the agarose into a gel tray with the well comb in place. Pour slowly to avoid bubbles that can disrupt the gel. Any bubbles can be pushed away from the well comb or towards the sides/edges of the gel with a pipette tip.
6. Let newly poured gel sit at room temperature until it has completely solidified.

Loading Samples and Running an Agarose Gel.

1. Add a loading buffer to each of your DNA samples.
2. Once solidified, place the agarose gel into the gel box (electrophoresis unit).
3. Fill the gel box with 1xTAE until the gel is covered.
4. Carefully load a molecular weight ladder into the first and last lane of the gel.
5. Carefully load your samples into the additional wells of the gel.
6. Run the gel at 100-120 V until the dye line is approximately 75-80% of the way down the gel. A typical run time is about 30-40 minutes, depending on the gel concentration and voltage.
7. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
8. Stain the gel with Ethydium Bromide on a rocker for 15-30 minutes.



CAUTION: Ethydium Bromide is a known mutagen. Wear a lab coat, eye protection and gloves when working with this chemical.

9. Using any device that has UV light, visualize the DNA bands. If you will be purifying the DNA for later use, use long-wavelength UV and expose for as little time as possible to minimize damage to the DNA.

Follow protocol of Cytiva GFX™ PCR DNA and Gel Band Purification Kit, as described below.

Consumables

• Cytiva GFX™ PCR DNA and Gel Band Purification Kit (Capture buffer type 3, Wash buffer type 1, GFX Microspin columns and 2mL collection tubes)
• MilliQ
• DNA sample to be purified/li>

Equipment

• Centrifuge
• Heating block

Protocol

Sample capture

1. Weigh empty 1.5mL Eppendorf tube.
2. Cut out band of interest from gel and place in Eppendorf tube.
3. Calculate weight of agarose gel slice.
4. Add 10 µL of Capture buffer type 3 for each 10 mg agarose gel slice. Mix by inversion.
5. Melt in heating block at 60 C until completely dissolved.
6. Check color of Capture buffer type 3-sample mix is yellow or pale orange.

Sample binding

1. Add 600 µL Capture buffer type 3-sample mix to assembled GFX Microspin column and collection tube.
2. Incubate 60 s at room temperature.
3. Spin 30 s at 16.000 × g in centrifuge. Discard flow through.
4. Place GFX MicroSpin column inside the same Collection tube.
5. Repeat until all sample is loaded.

Wash & dry

1. Add 500 μl Wash buffer type 1.
2. Spin twice 30 s 16 000 × g.
3. Discard Collection tube. Transfer GFX MicroSpin column t a clean 1.5ml microcentrifuge tube.

Elution

1. Add 30 μl MilliQ to MicroSpin column.
2. Incubate 60 s at room temperature.
3. Spin 60 s 16 000 × g.
4. Retain flow through.
5. Store purified sample DNA at -20°C.

Consumables

• Bacterial growth medium (suited to species)
• 80% glycerol
• Cryo-tube (2 per culture)
• Liquid culture of desired strain

Equipment

• Centrifuge
• Vortex

Protocol

Note: Take care when preparing glycerol stocks, since you will often use them for many years to come. Make sure to perform all steps in which the culture/glycerol stock is handled only at the sterile working bench.

1. Centrifuge bacterial culture in sterile Falcon tube (4000 g, 15 min, room temperature).
2. Discard the supernatant
3. Resuspend the cell pellet in 1.5 ml of a suitable medium.
4. Prepare two cryo tubes per culture by labeling them on the side and on the top.
5. Pipet 750 ul of resuspended cells into each of the cryo tubes.
6. Add 750 ul of sterile 80% glycerol into each of the cryo tubes.
7. Mix well by vortexing the closed cryo tubes.
8. Freeze the glycerol stocks in liquid nitrogen.
9. Deposit the glycerol stocks in a box in the -80° C freezer.

Consumables

• Sterile minimal medium with methanol
• M. extorquens colony from agar plate
• Appropriate antibiotic (1000x stock)

Equipment

• Erlenmeyer or culture tube
• Shaking incubator

Protocol

Note: work sterile in a flow cabinet.

• Add minimal medium with methanol to an erlenmeyer (use 1/3 of the total volume of the erlenmeyer) and if necessary, add the appropriate amount of needed antibiotic to reach a final concentration of:

• Inoculate a single M. extorquens colony from an agar plate to the medium.
• Incubate in a shaking incubator at 30 °C at 180 rpm.

Based on: H. Toyama et al. 1998 FEMS Microbiology Letters 166:1-7

Consumables

• Sterile water, cold
• Sterile 10% glycerol, cold
• Wildtype M. extorquens culture

Equipment

• Shaking incubator
• Centrifuge
• Liquid nitrogen

Protocol

1. Collect an exponentially growing culture of M. extorquens at OD 1.0 -1.5
2. Incubate 15 min on ice, then centrifuge at 4 °C, 4000 rpm for 15 min.
3. Washing: Resuspend pellet with 1 volume of cold sterile water, centrifuge at 4 °C, 4000 rpm for 15 min. Repeat the washing step with water.
4. Resuspend pellet with 1/2 volume of cold, sterile 10% glycerol solution, centrifuge at 4 °C, 4000 rpm for 15 min.
5. Resuspend cells in 1/100 of volume of cold, sterile 10% glycerol solution.
6. Make 50 µl aliquots, freeze with liquid nitrogen and store at -80 °C.

CAUTION: Liquid nitrogen is a simple asphyxiant and exists at cryogenic temperatures. Handle with skin protective equipment and never keep more than 500 ml of liquid nitrogen in a standard size room.

Based on: H. Toyama et al. 1998 FEMS Microbiology Letters 166:1-7

Consumables

• Methylobacterium nutrient broth
• Plasmid
• 2 mL Eppendorf tubes
• M. extorquens medium plates + antibiotics
• Competent M. extorquens (-80C in freezer)
• Plastic plate spreader (sterile)

Equipment

• Pipette + tips (p200-p20)
• Icebox
• Bunsenburner
• Electroporation machine + cuvette
• Shaking incubator

Protocol

1. Thaw competent cells on ice.
2. For suicide vectors: Add 1 µg of DNA (max. 2 µl of plasmid can be added). For replicating plasmids: Add 100-300 ng of DNA (max. 2 µl of plasmid can be added).
3. Mix cells and plasmids by flicking them.
4. Transfer cells to cold electroporation cuvettes (1 mm gap). Avoid bubbles by tapping firmly against a surface. Remove by pipette if necessary
5. Trigger the pulse – E. coli preset program (1.8 kV, 5 ms time constant).
6. Add 1 ml of Nutrient Broth and transfer cells to a sterile 2 ml Eppi.
7. Incubate with shaking at 30 °C for at least 3 hours.
8. Spread on plates: make 1000-fold dilution for replicating plasmids; plate 150 µl. Plate 150 µl undiluted & 1:10 dil. for suicide vectors.

Consumables

• Sterile LB medium
• E. coli colony from agar plate
• Appropriate antibiotic (1000x stock)

Equipment

• Erlenmeyer or culture tube
• Shaking incubator

Protocol

Note: work sterile in a flow cabinet.

• Add LB medium to an erlenmeyer or culture tube (use 1/3 of the total volume of the erlenmeyer or culture tube) and if necessary, add the appropriate amount of needed antibiotic to reach a final concentration of:

• Inoculate a single E. coli colony from an LB agar plate to medium.
• Incubate overnight in a shaking incubator at 37 °C at 200 rpm.

Consumables

• Solution A: 80 mM MgCl₂ + 20 mM CaCl₂ + ddH₂O (20 mL needed for 200 mL culture)
• 2.5 M MgCl₂, 5 M CaCl₂, 20% Glycerol retrieved from stock.
• Solution B: 100 mM CaCl₂ + 10% glycerol + ddH₂O (8mL needed for 200 mL culture)
• Ice-cold sterile 1.5 mL Eppendorf tubes
• Ice-cold sterile 50 mL falcon tubes
• Liquid nitrogen
• Exponentially growing E.coli (Top10) culture

Equipment

• Pre-chilled 4 °C centrifuge
• Cooler with ice
• Liquid nitrogen dewar
• Safety glasses
• Tweezer/extra long forceps

Protocol

1. Set up a small volume cell culture in a sterile glass flask in LB and incubate overnight in 37 °C shaker
2. Dilute overnight culture 1:100 cell culture in 200 mL LB, grow until OD600 = 0.6 - 1.0
3. Aliquot cell culture into 4 sterile 50 mL falcon tubes (pre-chilled) an put on ice 20 mins
4. Centrifuge at 4 °C degrees and 4000-5000 rpm for 10 mins (4000 rpm)
5. Keep cell pellet and discard supernatant.
6. Gently resuspend pelleted cells in each 50 mL falcon using 5 mL solution A (pre-chilled). Tip: merge the content of 2 falcons in one (total volume is 10 mL).
7. Centrifuge at 4 °C degrees, 4000 - 5000 rpm for 10 mins, discard supernatant and keep cell pellet (4000 rpm)
8. Resuspend each falcon with 4 mL Solution B (pre-chilled). Note: if you do not merge the content of 2 falcons as suggested in step 7 (i.e. because you're using 100 mL only), then resuspend.
9. Aliquot 50 µL competent cells in sterile pre-chilled Eppendorf tubes and flash freeze them in liquid nitrogen



CAUTION: Liquid nitrogen is a simple asphyxiant and exists at cryogenic temperatures. Handle with skin protective equipment and never keep more than 500 ml of liquid nitrogen in a standard size room.

10. Store them at -80 degrees.

Consumables

• LB agar plates with appropriate antibiotic
• Frozen Top10 E. coli competent cells
• Plasmid DNA
• LB medium
• Plastic plate spreader (sterile)
• Chemically competent E.coli

Equipment

• Heat block
• Incubator
• Ice box

Protocol

1. Incubate 1-2 µL plasmid DNA + 50 µL frozen Top10 E. coli competent cells on ice for 30 minutes.
2. Heatshock for 60 seconds at 42 °C.
3. Incubate 2 min on ice.
4. Add 500 µL LB medium.
5. Incubate at 37 °C for 1 hour.
6. Plate 100 µL of the plasmids on LB agar+antibiotic.
7. Incubate overnight at 37 °C or over the weekend on the bench (room temperature).

Follow protocol of NucleoSpin Plasmid EasyPure kit, as described below.

Consumables

• NucleoSpin Plasmid EasyPure kit
• 5 mL overnight liquid culture of transformed E. coli
• MilliQ
• 1.5 ml microcentrifuge tubes

Equipment

• Centrifuge
• Microcentrifuge
• Pipettes + pipette tips
• Nanodrop
• Freezer

Protocol

1. Centrifuge a 5 mL overnight liquid culture twice for 5 min at 3000 rpm.
2. Discard supernatant and transfer pellet into a microfuge tube.
3. Add 150 µL Buffer A1 and vortex to resuspend cells completely.
4. Add 250 µL Buffer A2 and invert 5 times.
5. Incubate up to 2 minutes at room temperature to lyse cells.
6. Add 350 µL Buffer A3 and invert until lysate has turned colorless.
7. Centrifuge for 3 min at full speed to pellet precipitate.
8. Put a NucleoSpin Plasmid EasyPure Column into a collection tube (2 mL).
9. Load clear supernatant onto the spin column.
10. Centrifuge for 30 sec at 3800 rpm and discard flow through.
11. Add 450 µL Buffer AQ to spin column.
12. Centrifuge for 1 min at full speed and discard collection tube.
13. Put spin column into a new 1.5 mL microcentrifuge tube.
14. Add 40 µL of MilliQ and incubate for 1 min.
15. Centrifuge for 1 min at full speed.
16. Measure concentration using nanodrop.
17. Keep isolated plasmids in freezer (-20 °C).

Based on: Sung won Lim, Sebastian Cocioba 2023. Quick and dirty sequencing microbial genome extraction v1. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1qjx7gr2/v1

Consumables

• MilliQ
• Tris
• EDTA
• 6M HCl
• SDS
• Sodium acetate trihydrate
• 70% EtOH
• Proteinase K
• RNase A
• Storage buffer of choice (usually milliQ)
• 1% agar
• TAE buffer
• DNA ladder
• DNA loading dye

Equipment

• Vortex
• Gel-electrophoresis set-up
• Nano-drop set-up
• Pipettes, pipette tips, flasks etc.
• Centrifuge
• Scale
• Spectrophotometer
• Refrigerator
• 37 °C shaker

Protocol

1. Prepare 100 mL Edward's buffer:

   a. Add 80 mL milli-Q H2O in (sterile) flask.

   b. Add 3.15 g of TRIS.

   c. Add 0.93 g of EDTA (disodium hydrate).

   d. Add 1.46 g of NaCl.

   e. Add 6M HCl until the solution is pH 8.

   f. Add milli-Q H2O until the solution is 100 mL.

   g. Add 0.5 g of SDS.

2. Prepare 20 mL of 3M (pH 5.4) sodium acetate:

   a. Add 20 mL of milli-Q H2O in (sterile) flask.

   b. Add 8.165 g of sodium acetate trihydrate.

   c. Add 6 M HCl until the solution is pH 5.4.

3. Prepare 20 mL of isopropyl alcohol (also known as 2-propanol).
4. Prepare 10 mL of 70% EtOH.
5. Centrifuge 20 mL of culture with OD600 of 0.8-1.5 resuspend pellet in 1 mL of M. extorquens standard medium on methanol.
6. Spin down 1 mL of culture and resuspend the pellet vigorously in 100µL of Edwards buffer.
7. Transfer to PCR-tube and add 2 µL of RNase A. Mix vigorously and vortex 10 seconds.
8. Incubate at 37 °C for 15 min.
9. Add 2 µL of Proteinase K and incubate at 55 °C for 60 min.
10. Deactivate enzymes by incubating at 95 °C for 10 min.
11. Transfer to 1.5 mL Eppendorf tube.
12. Add 10% 3 M (pH 5.4) sodium acetate, and 1:1 volume of 100% isopropyl alcohol. This is best done by transferring 90 µL to the Eppendorf tube in the previous step, adding 10 µL of sodium acetate and adding 100 µL of isopropyl alcohol.
13. Invert tube 10 times - precipitates should begin to form. Do not vortex and handle pellet carefully.
14. Spin down at max speed for 5 min.
15. Decant supernatant and resuspend pellet in 1 mL of 70% EtOH. Pellet will not dissolve.
16. Spin down at max speed for 5 min.
17. Repeat 70% EtOH resuspension and washing step at least 2 more times.
18. Decant completely and air dry for 5 minutes. Don't let the pellet overdry.
19. Resuspend in 100 µL milliQ, or storage buffer of choice.
20. Incubate in a 37 °C shaker for 60 minutes - the extract is likely to not be dissolved fully.
21. Incubate in 4 °C overnight. Depending on yield the solution will be extremely viscous.
22. Check quality using gel electrophoresis and concentration using nanodrop.

Follow protocol of manufacturer’s kit.

Consumables

• Primers (10 µM)
• MilliQ water
• Loading dye
• 1 kb ladder
• Eppendorf tubes
• PCR tubes
• Template DNA
• Q5 HF 2x Master mix
• Ethidium bromide
• TAE buffer
• 1% agarose

Equipment

• PCR machine
• Gel electrophoresis machine
• Gel doc

Protocol

1. Make a master mix (for all PCR reactions).

Per 1 PCR reaction:

	Component	Volume per reaction
1	Template DNA	1 µL
2	MilliQ	9 µL
3	Q5 HF 2x Master mix	12.5 µL

2. Fill PCR tubes with following components (Also include template only control or primer only control):

Per PCR reaction:

	Component	Volume per reaction
1	Forward primer (10 µM)	1.25 µL
2	Reverse primer (10 µM)	1.25 µL
3	Master mix	22.5 µL

3. Fill PCR tubes with following components (see table):
4. Run PCR program (use T_a of used primer pair).

PCR program:

	Step	Program	Temperature	Unit	Duration	Unit
1		Initial Denaturation	98	°C	30	sec
2	3 steps (30-35 cycles)	Denaturation	98	°C	5-10	sec
3		Annealing	Ta	°C	10-30	sec
4		Extension	72	°C	20-30	sec/kb
5		Final Extension	72	°C	2	min

5. Run gel electrophoresis with the following products:

a. Ladder.

b. PCR products with loading dye.

6. Run for 40 mins at 120 V.

Follow protocol of manufacturer’s kit.

Consumables

• MilliQ
• PCR tubes
• GoTaq polymerase PCR kit (GO Taq polymerase, GoTaq buffer (with or without loading dye)
• dNTPs
• Primers (10 µM)
• Template DNA/Colony of interest
• 1 kb DNA ladder
• Eppendorf tubes
• Ethidium bromide
• TAE buffer
• 1% agarose gel

Equipment

• Thermocycler
• Pipettes
• Gel electrophoresis machine
• Gel doc gel photography machine

Protocol

1. Resuspend colonies in 5µL MillliQ in PCR tubes. Plate 4 µL on a new agar plate with appropriate antibiotics. Use 1 µL as template for PCR reaction.
2. Prepare master mix (store stock in -20 freezer).

Master mix for 200 reactions:

	Component	Volume
1	GoTaq buffer (5x)	1000 µL
2	dNTPs	100 µL
3	GO Taq polymerase	25 µL

3. Prepare the mix for all PCR tubes (+ some extra). Also include no template control, no primer control and optionally a positive control.

Per PCR reaction:

	Component	Volume
1	Master Mix	5.625 µL
2	Fwd primer (10 µM)	1.25 µL
3	Rev primer (10 µM)	1.25 µL
4	MilliQ	15.875 µL

4. Add 24 µL of PCR mix with 1 µL of the target DNA/suspended colony.
5. Run PCR (30 - 35 cycles).

PCR program:

	Step	Program	Temperature	Unit	Duration	Unit
1		Initial Denaturation	95	°C	2	min
2	3 steps (30-35 cycles)	Denaturation	95	°C	30-60	sec
3		Annealing	Ta	°C	30-60	sec
4		Extension	73	°C	1	min/kb
5		Final Extension	73	°C	5	min

6. Run gel electrophoresis with 5 µL ladder and 5 µL of the PCR products + 1 µL loading dye. If GoTaq mix already contains loading dye, load 6 µL sample directly onto the gel.
7. Stain 10-30 min with ethidium bromide and scan with gel doc.

Based on manufacturer’s protocol.

Consumables

• Appropriate restriction enzyme
• Vector/plasmid
• 10x rCutSmart buffer
• MilliQ
• 1.5 mL Eppendorf tube

Equipment

• Incubator

Protocol

1. Set up the reaction on ice in a 1.5 mL Eppendorf tube

Reaction component	Amount	Note
Vector/plasmid	1 µg	Calculate the needed volume based on the concentration
rCutSmart buffer (10x)	5 µL
Restriction enzyme	1 µL	Choose appropriate restriction enzyme(s)
MilliQ	To 50 µL

2. Incubate at 37 °C. When using a high fidelity restriction enzyme, incubate for 1 hour, otherwise for 3 hours.
3. After incubation, check if the whole sample is digested using gel electrophoresis.
4. If digestion results in multiple DNA fragments, perform gel purification.

Based on manufacturer’s protocol.

Consumables

• Digested plasmid/vector
• Insert(s)
• T4 DNA ligase
• T4 DNA ligase buffer

Equipment

• Heating block or incubator

Protocol

1. Set up the reaction on ice in a 1.5 mL Eppendorf tube. Add ligase last. Gently mix by pipetting up and down and microcentrifuge shortly.

Reaction component	Amount	Note
Digested Vector/plasmid		Calculate the needed volume based on the concentration
Insert(s)		Calculate the needed volume based on the concentration
T4 DNA ligase buffer
T4 DNA ligase
MilliQ	To 10 µL

2. Incubate at 30 °C for 10 min. Heat inactivate it at 65 °C for 10 min.
3. Chill on ice before transformation.

Follow manufacturer’s protocol.

Consumables

• NEB HiFi Assembly Master Mix (2X)
• Vector
• Insert(s)
• NEB positive control reaction mix
• MilliQ
• Top10 competent E. coli

Equipment

• Thermocycler
• Ice box
• Incubator

Protocol

General overview of the process

1. Design primers to amplify fragments (and/or vector) with appropriate overlaps.
2. PCR amplify fragments using a high-fidelity DNA polymerase (Q5).
3. Prepare linearized vector by PCR amplification using a high-fidelity DNA polymerase or by restriction digestion.
4. Confirm and determine concentration of fragments using agarose gel electrophoresis, a Nanodrop™ instrument or other method.
5. Add fragments and linearized vector to Gibson Assembly Master Mix and incubate at 50 °C for 15 minutes to 1 hour, depending on number of fragments being assembled.
6. Transform into Top10 Competent E. coli.

Assembly protocol

1. Use the provided table to calculate needed volumes of vector and inserts. Insert bp length, concentration, molar ratio of inserts to vector, moles and total volume. Use 5 µL for total volume and 2.5 µL of 2x HiFi assembly master mix.
2. Adjust molar ratio and/or amount if needed.

Component	Concentration of sample	Ratio	pmol	ng	μL
Insert	in ng/μL	5	molar ratio * amount of vector	pmol * bp * 650 / 1000	mass/concentration
Vector	in ng/μL	0.01	molar ratio * amount of vector	pmol * bp * 650 / 1000	mass/concentration

3. Set up the following reaction on ice. Also include a positive control sample using 1:1 NEB positive control reaction mix and HiFi Assembly Master Mix (2X).

Component	Volume
Insert 1	[Calculate using table]
Vector	[Calculate using table]
HiFi Assembly Master Mix (2X)	2.5 µL
MilliQ	2.5 µL - volume of fragments
Total volume	5 µL

4. Incubate samples in a thermocycler at 50 °C for 1 hour. Following incubation, store samples on ice or at –20 °C for subsequent transformation.
5. Transform Top10 Competent E. coli cells with 2.5 µL of the assembly reaction, following the Top10 transformation protocol.

Protocol based on: R. Ledermann, S. Strebel, C. Kampik, and H. M. Fischer, “Versatile vectors for efficient mutagenesis of Bradyrhizobium diazoefficiens and other alphaproteobacteria,” Appl. Environ. Microbiol., vol. 82, no. 9, pp. 2791–2799, May 2016, doi: 10.1128/AEM.04085-15.

Protocol

1. Prepare sufficient amounts of pREDSIX and pRGD-KmR from a transformed TOP10 E. coli culture.
2. Amplify the upstream and downstream regions of the gene (750 bp each) by PCR using the genome of M. extorquens as template DNA. Introduce an NdeI restriction site in one of the primers so that it sits between the upstream and downstream regions.
3. Digest pREDSIX with MfeI, with dephosphorylation, and purify the digested vector.
4. Do Gibson assembly with the digested pREDSIX, upstream and downstream flanking regions.
5. Transform into TOP10 E. coli. Use ampicillin as antibiotic selection. Confirmation successful Gibson assembly by colony PCR. Isolate plasmid from liquid culture and confirm successful Gibson assembly with sequencing as well.
6. Digest this vector with NdeI, with dephosphorylation, and purify the digested vector.
7. Digest pRGD-KmR with NdeI. Use at least 2 µg of vector. Run the digested vector on gel. Isolate band that is 1228 bp, which is the KmR cassette. Purify this gel band.
8. Ligate KmR cassette into digested pREDSIX with flanks.
9. Transform into TOP10 E. coli. Use ampicillin and kanamycin as antibiotic selection. Confirm successful ligation by colony PCR. Isolate the plasmid from liquid culture and confirm successful ligation with sequencing as well.
10. Transform the complete pREDSIX with flanks and KmR cassette into M. extorquens by electroporation. Use kanamycin as antibiotic selection.
11. Select colonies without mCherry expression using fluorescence microscopy.
12. Validate the gene knockout by PCR with primers outside the flanking regions using genome of the mutant strain as template DNA and compare to wild-type M. extorquens.

Based on: Zuriani, R., Vigneswari, S., Azizan, M.N.M. et al. A high throughput Nile red fluorescence method for rapid quantification of intracellular bacterial polyhydroxyalkanoates. Biotechnol Bioproc E 18, 472–478 (2013). https://doi.org/10.1007/s12257-012-0607-z

Consumables

• MilliQ
• Nile Red Stock (80 µg/ml Nile Red in DMSO)
• DMSO
• Aluminum foil

Equipment

• Pipette
• Centrifuge
• Vortexer
• 96-well plate reader

Protocol

1. Centrifuge 1 mL cell suspension (Cell concentration 0.5-1.0mg/mL) (12000g, 5min) (Ideally use 0.8mg/mL cell concentrations).
2. Discard supernatant and resuspend in 1 mL MilliQ.
3. Add 40 µL Nile Red stock to solution (80µg/mL in DMSO; 3.1µg/mL suspension).
4. Incubate at room temperature for 30min.
5. Centrifuge (12000 g, 5min)
6. Discard supernatant and resuspend pellet in 1 mL MilliQ.
7. Vortex vigorously until fully resuspended.
8. Aliquot into 96-well plate (200µL).
9. Measure fluorescence.
10. Load 96-well plate into plate reader.

    a. Excitation= 535nm

    b. Emission= 605nm

11. Determine PHA concentration from nile red fluorescence intensity using a PHA calibration curve.

Based on: Mayooreshwar P Rajankar, Sapna Ravindranathan, P R Rajamohanan, Anu Raghunathan, Absolute quantitation of poly(R)-3-hydroxybutyric acid using spectrofluorometry in recombinant Escherichia coli, Biology Methods and Protocols, Volume 3, Issue 1, 2018, bpy007, https://doi.org/10.1093/biomethods/bpy007

Consumables

• 0.1 M glycine-HCL buffer pH 3.0
• 50 ml falcon tube
• Standard PHA (Sigma cat no. 363502)

Equipment

• Fluorometer with LED light source
• LED light source in raw mode for excitation.

  - Excitation 535 nm

  - Slits fixed at 20 nm

  - Emission 600-640 nm

• Ice bath
• Sonication chamber

Protocol

1. Prepare the standard PHB suspensions of 1000 µg/mL by suspending 10 mg of PHB (Sigma: cat no. 363502) in 10 mL of 0.1 M glycine-HCl buffer pH 3.0 in 30 mL Borosil flat bottom culture tube (Cat no. 9910010).
2. Incubate the suspension in ice bath for 20 min.
3. Probe sonicated the suspension in ice bath at 40% amplitude for 10 min at a 10/20 cycle on Sonic Vibracell VCX130( 130 wattz, 20 kHz) equipped with 6 mm stepped tip sonication probe.
4. Add to 1µg/mL Nile red (Sigma: cat no. N3013) (in DMSO 0.5 mg/mL) to sonicated standard suspensions and incubated for 2h at room temperature (25 °C).
5. Dilute Nile red stained sonicated PHB suspensions with 0.1 M glycine-HCl buffer to 1 mL in the conc. range of 20 to 1000 µg/mL)
6. Aliquot 200 µL suspensions at various concentrations (in the range of 20 to 1000 µg/mL)
7. Wash 200 µL Nile red stained sonicated granules twice with rigorous vortexing in 200 µL 0.1 M glycine-HCl buffer followed by centrifugation* at 12000 rpm for 5 min.
8. Discard the supernatant buffer and add 200 µL ethanol to release the Nile red bound to the PHB standard granules.
9. Measure the fluorescence from ethanol extract on Qubit 2.0 Fluorometer (Thermo Fisher Scientific; Cat no. Q32866) with blue LED light source in raw mode.



Note: All the centrifugation steps should be carried out at room temperature.

Consumables

• 96-wellsplate(Thermo Fisher Scientific-Nunclon 96 Flat Bottom Black Polystyrene Catalog No.: 137101/137103/237105/237107/237108/437111/437112)
• >M. extorquens minimal medium
• Inducers

Equipment

• Plate reader (Tecan i-control infinite 200Pro)

Protocol

1. Grow the Methylobacterium strains of interest in culture medium (minimal medium with MeOH) until saturation.
2. Prepare the different media you need for your growth experiment (carbon sources).
3. Spin down at 4000 rpm for 10 minutes at 4 °﮲C and remove supernatant.
4. Resuspend the cells in 2 mL Minimal medium.
5. Dilute the concentrated cell suspension 50x in a cuvet (20 µL cell suspension + 980 µL H₂0).
6. Measure the absorption at 600 nm.
7. Calculate the real OD600 by using this formula:
   OD600 = (absorption in well - 0.0755)/0.2344
8. Calculate how many µL of cell suspension you need to add to 1 mL to get the desired starting OD600, 0.05.
9. Calculate and prepare inducer solutions, stock should be 1000x end concentration. See end concentration below:
   - Vanillic acid¹ : 5, 10, 25, 50, 75, 100, 150, 200, 250 µM dissolved in ethanol
   - Cumate² : 1, 2, 5, 10, 15, 20, 30, 50, 100 µM dissolved in water
   - IPTG³ : 5, 10, 25, 50, 100,, 150, 250, 500, 1000 µM dissolved in water
10. Prepare all the strain and media combinations in 2 mL Eppendorf tubes (1 mL medium + x µL bacteria + 1 µL inducer if necessary (for IPTG highest concentration, 2 microliter))
11. Vortex before adding 200 µL cell suspension per well in the 96-wells plate.
12. Seal the plate with parafilm and put it in the Tecan plate reader.
13. Use the appropriate script and start the experiment.

Mode	Absorbance
Measurement Wavelength	600
Bandwidth	9
Number of Flashes	25
Settle Time	0
Mode	Fluorescence Top Reading
Excitation Wavelength	575
Emission Wavelength	610
Excitation Bandwidth	9
Emission Bandwidth	20
Gain	100
Number of Flashes	25
Integration Time	20
Lag Time	0
Settle Time	0
Z-Position (Manual)	20000

References

1. Kaczmarczyk A, Vorholt JA, Francez‐Charlot A. Synthetic vanillate-regulated promoter for graded gene expression in Sphingomonas. Scientific Reports. 2014;4(1). doi:10.1038/srep06453
2. Kaczmarczyk A, Vorholt JA, Francez‐Charlot A. Cumate-Inducible gene expression system for sphingomonads and other alphaproteobacteria. Applied and Environmental Microbiology. 2013;79(21):6795-6802. doi:10.1128/aem.02296-13
3. Carrillo M, Wagner M, Petit F, Dransfeld A, Becker A, Erb TJ. Design and control of extrachromosomal elements in Methylorubrum extorquens AM1. ACS Synthetic Biology. 2019;8(11):2451-2456. doi:10.1021/acssynbio.9b00220

Consumables

• CHCA, α-cyano-4-hydroxycinnamic acid
• ACN, Acetonitrile
• Water
• TFA, Trifluoroacetic acid
• Methanol, 90%
• Soft, lint-free paper wipes
• Formic acid/methanoic acid
• Target bacterial colonies

Equipment

• MALDI-TOF
• 48 spot steel target MALDI plate
• Fume hood

Protocol

Preparation of MALDI matrix

1. Prepare 10 mg/mL MALDI-grade, and/or recrystallized α-cyano-4-hydroxycinnamic acid (CHCA) in MS-grade solvents: 50% acetonitrile (ACN), 47.5% water (H2O), 2.5% trifluoroacetic acid (TFA). Example: 100 μL solution = 50 μL ACN + 47.5 μL H2O + 2.5 μL TFA + 1 mg CHCA
2. Prepare at least 1 μL of matrix solution per MALDI plate spot and vortex or sonicate until in solution (approximately 5 min sonication or no visible solids).



CAUTION: TFA is a strong acid that should be handled in a chemical fume hood while wearing proper personal protective equipment, as it can damage skin, eyes, and airways with contact or inhalation.

Preparation of MALDI target plates

1. Rinse MALDI plate with methanol (HPLC-grade or higher) and wipe dry with soft paper wipes. Do not use abrasive brushes when cleaning target plates, as this can permanently damage the surface of the target plate.
2. Assign spots:

   a. Assign protein and specialized metabolite calibrant spots.

   b. Organize calibration spots evenly across the sample population, to account for MALDI-plate-irregularities and instrument drift over time.

3. Using a sterile toothpick or pipet 0.5 μL transfer a small portion of a bacterial colony/prepared bacterial sample to the appropriate spot on the MALDI plate. Spread the bacterial colony evenly over the spot. The spot should appear as flat as possible.



NOTE: It will be easier to flatten bacterial colonies that are more mucoid/amorphous. For more rigid/solid colonies, avoid leaving visible clusters of cell mass on the MALDI spot.

4. Overlay 1 μL of 70% mass spectrometry grade formic acid onto each sample spot. Allow acid to air dry completely in a chemical fume hood (approximately 5 min).
   

   CAUTION: Formic acid is a caustic chemical and should be handled in chemical fume hoods. It can damage airways if inhaled.

5. Add 1 μL of the prepared MALDI matrix solution to each sample spot, as well as to the matrix/media control spots. Allow matrix solution to air dry completely (approximately 5 min).
6. Seek assistance in operating MALDI-TOF spectrometers. Operation requires certification.

Consumables

• MilliQ
• Nile Red Stock (80µg/ml Nile Red in DMSO)
• DMSO
• 3% analytical Agarose
• Microscopy slides + coverslip
• Aluminum foil

Equipment

• Pipette
• Centrifuge
• Vortexer
• Fluorescence microscope + filter (Excitation= 535nm, Emission= 605nm)

Protocol

Nile red staining of cells

1. Centrifuge 1mL cell suspension (Cell concentration 0.5-1.0mg/mL) at 12000g 5min (Ideally use 0.8mg/mL cell concentrations).
2. Discard supernatant and resuspend in 1mL MilliQ.
3. Add 40µL nile red stock to solution (80µg/mL in DMSO; ==> 3.1µg/mL suspension).
4. Incubate at room temp. 1 hour.
5. Centrifuge @12000g 5min.
6. Discard supernatant and resuspend pellet in 1mL MilliQ.
7. Vortex vigorously until fully resuspended.
8. Keep in cool dark space, wrapped in reflective foil.

Preparation of microscopy slide

1. Prapare 3% analytical agarose and keep as hot as possible.
2. Thoroughly clean one side of microscope slide with clean cloth.
3. Using pipette, place droplet of liquid agarose on cleaned size of slide, then press coverslip onto droplet as quickly as possible.
4. Allow to set.

Observe under microscope

1. Remove coverslip from flattened agarose.
2. Load 10μl of stained cell suspension onto slide and replace cover slip.
3. Under transmitted full spectrum light, identify regions with large number of cells visible through ocular (50+).
4. Set consistent exposure time for photos (200 ms - 400ms recommended).
5. Create multilayer photo of transmitted light and fluorescence emission.

Consumables

• 800 mL of M. extorquens Minimal Medium on Methanol
• Precultures of Methylobacterium extorquens AM1 to inoculate
• 100 mL of PBS

Equipment

• 60x 2 mL Eppendorf tubes
• 60x 15mL falcon tubes
• Pipette tips, pipette, 5mL pipette tips, pipetboy
• Stove
• Analytical balance
• Gloves
• Spectrophotometer

Protocol

1. Inoculate M. extorquens in M. extorquens Minimal Medium at OD = 0.1, using your M. extorquens pre cultures. Make 500mL M. extorquens for the OD=2 calibration and 300 mL of M. extorquens for the OD600 = 4 culture. Do this in the afternoon of day 0.
2. Put 30 2mL Eppendorf tubes overnight in the stove at 100 °C. Do this in the evening of day 0.
3. Label and weigh the Eppendorf tubes in the morning of day 1 three times on the analytical balance. Weigh in triplicate, use gloves.
4. Measure OD600 of the 500mL culture after 20 hours, this is the morning of day-1, in quintuplicate (5x) using a 1-10 dilution. Calculate back to the real OD600 (ODreal = OD measured x 10), and concentrate culture so that it will become a OD600 = 0 culture of at least a 100mL. Dilute down to 3x10 cultures of OD600 = 1, 2, ...., 9, 10. Make sure each culture consists of at least 5mL.
5. Spin down 5mL of all cultures, resuspend pellets in 500µL of PBS and transfer to the pre-weighted labeled 2 mL Eppendorf tubes.
6. Remove supernatant (centrifuge 4000 g 10min) and dry the 2mL Eppendorf tubes with pellet overnight at 100 °C.
7. Weigh each Eppendorf in triplicate on day 2 afternoon, use gloves. Calculate cell dry weight (CDW), divide by 5mL to get CDW/mL.
8. Measure on the morning of day 2 the OD600 of the 300mL culture. This should be in between OD600 = 3.2 & 4.5. Repeat steps above for the 300 mL culture.

Based on: Mongili, B., Abdel Azim, A., Fraterrigo Garofalo, S. et al. Novel insights in dimethyl carbonate-based extraction of polyhydroxybutyrate (PHB). Biotechnol Biofuels 14, 13 (2021). https://doi.org/10.1186/s13068-020-01849-y

Consumables

• Chloroform
• Hexane (95%)
• H₂O
• Cell culture

Equipment

• Vacuum pump
• (Large) centrifuge
• Fridge
• Paper filter
• Glass bottles, pipettes etc.
• Oven
• Ceramic mortar and pestle

Protocol

1. Cultivate biomass using the cultivation protocol.
2. Collect the cell pellet by centrifugation by centrifuging 2x 10min at 4000rpm. Wash with H2O to collect pellet.
3. Dry the cell pellet for 12h at 105 °C, and ground by ceramic mortar and pestle.
4. Add chloroform to the pellet in a 15:1 chloroform:pellet (volumetric) ratio and stir 120 min at 60 °C.



WARNING: Chloroform is an acute respiratory irritant and known carcinogen! Use appropriate protective equipment (fume hood, double thick gloves) and minimise exposure times where possible.

5. Centrifuge for 5-10 minutes at 4000rpm and remove the pellet.
6. Add hexane in 1:3 hexane:chloroform ratio.



WARNING: 95% Hexane is highly combustible and a suspected carcinogen. Handle with care.

7. Leave the mix 24h at 4 °C and separate the precipitated PHA by vacuum filtration.
8. Air dry the PHA on filter paper.
9. Quantify the PHA.

Based on: Mongili, B., Abdel Azim, A., Fraterrigo Garofalo, S. et al. Novel insights in dimethyl carbonate-based extraction of polyhydroxybutyrate (PHB). Biotechnol Biofuels 14, 13 (2021). https://doi.org/10.1186/s13068-020-01849-y

Consumables

• DMC (Dimethyl carbonate) (99%)
• Ethanol Anhydrous
• H₂O
• Cell culture

Equipment

• Vacuum pump
• (Large) centrifuge
• Fridge
• Paper filter
• Glassware, pipettes etc.
• Reflux column
• Fume hood
• Safety gloves

Protocol

1. Cultivate biomass according to cultivation protocol.
2. Collect cell pellets in a 50 mL falcon tube by centrifugation twice for 10 min at 4000 rpm. Wash with H2O and collect pellets.
3. Add 12 mL of DMC to the pellet, resuspend pellet and immediately transfer mixture to a 100 mL round glass flask which will later be used with the reflux set-up. Perform steps while wearing safety gloves and safety glasses and work in a fume hood.
4. Add a reflux column with cooling water onto the glass flask inside the fume hood (figure 1) and heat mixture to 85 degrees for 90 minutes while stirring at 700 rpm.
5. Let mixture cool to room temperature.
6. Add mixture to 50 mL falcon tubes, and centrifuge at 4000 rpm for 10 minutes.
7. Transfer organic phase to new 50 mL falcon tubes and add ethanol anhydrous in a 3:1 ethanol : DMC ratio to enable PHA precipitation. Store overnight at 4 °C.
8. Filter mixture with precipitated PHA through Wattmann filter paper #4 while wearing gloves.
9. Air-dry the paper overnight.
10. Quantify the PHA.

Protocol based on: Mongili, B., Abdel Azim, A., Fraterrigo Garofalo, S. et al. Novel insights in dimethyl carbonate-based extraction of polyhydroxybutyrate (PHB). Biotechnol Biofuels 14, 13 (2021). https://doi.org/10.1186/s13068-020-01849-y

Consumables

• Buffer pH 7.1
• Mineral salt solution
• Sterile methanol
• Iron solution
• Trace element solution
• 3% agarose
• 1-Butanol 99.9%
• Appropriate antibiotic
• IPTG (500 mM)
• Ethanol Anhydrous

Consumables

• M. extorquens strains of interest

Equipment

• Pipet tips, pipets, pipet boy
• Downflow cabinet
• Sterile 250 mL flasks

Protocol

1. Medium preparation: work sterile in downflow cabinet, add the following chemicals to 250 mL sterile flasks.
   • 30 mL buffer pH 7.1
   • 20 mL mineral salts
   • 500 µL methanol
   • 200 µL iron solution
   • 200 µL TE solution

2. Preparation of plates without butanol for the samples without pre-exposure to butanol.
   • Heat 1 bottle of 50 mL of agarose and add to the first flask.
   • Add 100 µL of Tetracyclin (10 mg/mL), leading to a concentration of 10 µg/mL.
   • Pour 1 plate with the first 50 mL in the flask.
   • Add 100 µL of IPTG to remaining ~50mL for a final concentration of 1 mM.
   • Pour 1 plate with the remaining ~50 mL in the flask, leave all plates to dry and turn them upside down.

3. Preparation of plates without butanol for the butanol pre-exposed samples.
   • Heat 1 bottle of 50 mL of agarose and add to the second flask.
   • Add 100 µL of Tetracyclin (10mg/mL), leading to a concentration of 10 µg/mL.
   • Pour 1 plate with the 50mL in the flask.
   • Add 100 µL of IPTG to remaining ~50 mL for a final concentration of 1 mM.
   • Pour 1 plate with the remaining ~50mL in the flask, leave all plates to dry and turn them upside down.

4. Preparation of plates with 0.25% butanol for the butanol pre-exposed samples.
   • Heat 50 mL agarose bottle and add to the third flask.
   • Add 250 µL 1-butanol for a concentration of 0.25% v/v
   • Add 100 µL of Tetracyclin (10mg/mL), leading to a concentration of 10 µg/mL.
   • Pour 1 plate with the 50mL in the flask.
   • Add 100 µL of IPTG to remaining ~50ml for a final concentration of 1 mM.
   • Pour 1 plate with the remaining ~50mL in the flask, leave all plates to dry and turn them upside down.

5. Preparation of plates with 0.5% butanol for the butanol pre-exposed samples.
   • Heat 50 mL agarose bottle and add to the fourth flask.
   • Add 500 µL 1-butanol for a concentration of 0.5% v/v
   • Add 100 µL of Tetracyclin (10 mg/mL), leading to a concentration of 10 µg/mL.
   • Pour 1 plate with the 50 mL in the flask.
   • Add 100 µL of IPTG to remaining ~50 mL for a final concentration of 1 mM.
   • Pour 1 plate with the remaining ~50 mL in the flask, leave all plates to dry and turn them upside down.

6. Preparation of plates with 0.75% butanol for the butanol pre-exposed samples.
   • Heat 50 mL agarose bottle and add to the fifth flask.
   • Add 750 µL 1-butanol for a concentration of 0.75% v/v
   • Add 100 µL of Tetracyclin (10 mg/mL), leading to a concentration of 10 µg/mL.
   • Pour 1 plate with the 50 mL in the flask.
   • Add 100 µL of IPTG to remaining ~50 mL for a final concentration of 1 mM.
   • Pour 1 plate with the remaining ~50 mL in the flask, leave all plates to dry and turn them upside down.

7. Butanol pre-exposure and plating.
   • Suspend a colony from each plate in 500 µL MiliQ water
   • Take 100 µL from each of these and pipet into epps
   • Add 2 µL 1-Butanol to 400 µL colony suspensions for 0.5% v/v
   • Mix and let sit on bench top for 30 min
   • Take 1 µL and drop onto plates cotton in triplo for each concentration - do dots
   • Incubate at 37 degrees Celsius till observable colonies.
   • Take pictures of plates and measure gray scale value of colonies using ImageJ. Normalize to background gray scale value.