Measure all components and dissolve them with approx. half of the total amount of water in a cylinder using a stir-fish. Prepare a Glucose stock solution (4%) in a separate cylinder. For 500 ml medium, use 50 ml of a 40% Glucose solution (= 20 g Glucose in 50 ml H2O).
Fill the medium solution up with demin. H2O (to 450 ml in case of 500 ml total, due to addition of Glucose later on) and pour the medium (for 500 ml) in a 1 l and the Glucose solution in a 500 ml bottle.
Gass them with N2 (2 cannulas, 30 min for 500 ml medium; 1 cannula, 20 min for 100 ml glucose). Measure and note down the pH value of the medium, then gas it for another 1 min with N2 before closing the bottle with a plug and lid.
Autoclave both bottles at 121°C for 20 min.
Using a cannula, add 50 ml of a 40% Glucose solution to 500 ml medium.
Using a 5 ml syringe, take out 5 ml of the medium into a 15 ml falcon to measure the pH value. A pH value of ~ 6.60 is desired.

Clostridial growth medium (CGM):

Measure all components and dissolve them with approx. half of the total amount of water in a cylinder using a stir-fish. Prepare a Glucose stock solution (4%) in a separate cylinder. For 500 ml medium, use 50 ml of a 40% Glucose solution (= 20 g Glucose in 50 ml H2O).
Fill the medium solution up with demin. H2O (to 450 ml in case of 500 ml total, due to addition of Glucose later on) and pour the medium (for 500 ml) in a 1 l and the Glucose solution in a 500 ml bottle.
Gass them with N2 (2 cannulas, 30 min for 500 ml medium; 1 cannula, 20 min for 100 ml glucose). Measure and note down the pH value of the medium, then gas it for another 1 min with N2 before closing the bottle with a plug and lid.
Autoclave both bottles at 121°C for 20 min.
Using a cannula, add 50 ml of a 40% Glucose solution to 500 ml medium.
Using a 5 ml syringe, take out 5 ml of the medium into a 15 ml falcon to measure the pH value. A pH value of ~ 6.60 is desired.

TYA medium:

Measure all components and dissolve them with approx. half of the total amount of water in a cylinder using a stir-fish. Prepare a Glucose stock solution (40%) in a separate cylinder. For 500 ml medium, use 50 ml of a 40% Glucose solution (= 20 g Glucose in 50 ml H2O). Fill the medium solution up with demin. H2O (to 450 ml in case of 500 ml total, due to addition of Glucose later on) and pour the medium (for 500 ml) in a 1 l and the Glucose solution in a 500 ml flask. Gass them with N2 (2 cannulas, 30 min for 500 ml medium; 1 cannula, 20 min for 100 ml glucose). Measure and note down the pH value of the medium, then gas it for another 1 min with N2 before closing the flask with a plug and lid. Autoclave both flasks at 121°C for 20 min. Using a syyringe, add 50 ml of a 40% Glucose solution to 500 ml medium. Using a 5 ml syringe, take out 5 ml of the medium into a 15 ml falcon to measure the pH value. A pH value of ~ 6.60 is desired.

54b Clostridium sp. medium

Dissolve ingredients (note that most of the carbonate remains suspended) and sparge medium with 100% N2 gas for 30 -45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials while stirring to keep carbonate suspended and autoclave.

135 Acetobacterium medium:

Dissolve ingredients except bicarbonate, fructose, vitamins, cysteine and sulfide, bring to the boil and cool to room temperature under 80% N2 and 20% CO2 gas mixture. Add bicarbonate (solid) and equilibrate the medium with the gas until a pH of around 7.4 is reached. Then distribute under the same gas atmosphere in anoxic Hungate-type tubes or serum vials and autoclave. Before use adjust the pH to 8.0 - 8.2 by adding a sterile anoxic stock solution of sodium carbonate (5% w/v) prepared under 80% N2 and 20% CO2 gas mixture (c. 0.25 ml per 10 ml medium) and add fructose, vitamins (sterilized by filtration), cysteine and sulfide from anoxic sterile stock solutions prepared under 100% N2. Note: For autotrophic growth D-fructose is omitted and a gas atmosphere of 80% H2 and 20% CO2 is used

YTF medium:


879 Clostridium ljungdahlii medium:

Dissolve ingredients (except bicarbonate, fructose, vitamins and reducing agents), sparge medium with 80% N2 and 20% CO2 gas mixture for 30 – 45 min to make it anoxic and adjust pH to 5.5. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave at 121°C for 15 min. Add fructose, vitamins (sterilized by filtration), cysteine and sulfide from sterile stock solutions prepared under 100% N2 gas atmosphere and bicarbonate from a sterile stock solution prepared under 80% N2 and 20% CO2 gas atmosphere. Adjust pH of complete medium to 5.9!

LB medium:


M9 medium:

Autoclave MgSO4 and CaCl2 seperately.

SOB medium:

Add MgCl2 x 6 H2O and MgSO4 x 7 H2O directly before use in autoclaved medium.

SOC medium:

Add components, except MgCl2 x 6 H2O solution (1 M), MgSO4 x 7 H2O solution (1 M) and glucose solution (1 M) to ddH2O and mix thoroughly. Adjust pH to 7.0. Bring volume to 960.0 ml. Autoclave for 20 min at 121 °C. Aseptically add sterile MgCl2 · 6 H2O solution (1 M), sterile MgSO4 · 7 H2O solution (1 M) and sterile glucose solution (1 M). Mix thoroughly.

TB medium:

Adjust pH of dissolved Pipes, CaCl2 and KCl solution to 6.7 with 2M NaOH. Autoclave dry MnCl2 separately.



These are the solutions/additives we used: Wolin‘s vitamin Solution:


Modified Wolin‘s vitamin solution:

First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH.

Amino Acid-Mix I:


Amino Acid-Mix II:

To dissolve L-Tyrosine adjust pH to 11.0.



These are the buffers we used: 50x TAE:


EPB S:


EPB NS:

These is the list of primers that we used:

- All backbones we used:

- All plasmids we created with new inserts:

E. coli
- DH5-alpha: 37 °C, LB medium
- XL-1 blue MRF: 37 °C, LB or M9 medium
- TOP10: 37 °C, LB medium
- CopyCutter EPI400: 37 °C in LB or SOC medium

Clostridium saccharoperbutylacetonicum ATCC 27021
30 °C in TYA, XY or CGM medium; N2-gassed anaerobic hungates/flasks or anaerobic agar plate

Clostridium beijerinckii ATCC 51743
37 °C in TYA, XY or 54b medium; N2-gassed anaerobic hungates/flasks or anaerobic agar plate

Clostridium beijerinckii NRRL B-593
30 °C in TYA, XY or 54b Clostridium sp. medium in N2-gassed anaerobic hungates/flasks or anaerobic agar plate

Clostridium ljungdahlii ATCC 55383
30 °C in XY or YTF medium; N2-gassed anaerobic hungates/flasks or anaerobic agar plate

Acetobacterium woodii ATCC 29683
30 °C in TYA, XY medium pH 8.0 or 135 Acetobacterium medium; N2-gassed anaerobic hungates/flasks or anaerobic agar plate

Fusion PCR
Fusion PCR was used to amplify our wanted inserts out of Clostridium ljungdhalii and Acetobacterium woodii. It was performed according to Harrison et al.
Pipetting scheme:


PCR program:




16S-rRNA DreamTaq-PCR
We performed 16S-rRNA PCR to check if we have pure cultures which are not contaminated.
Pipetting scheme:



PCR program:



PCR clean-up
We cleaned- up our insert PCR samples to use for digestion with the Qiagen QIAquick PCR purification Kit according to the manufacturer's protocol.

gDNA isolation
We isolated gDNA from Clostridium ljungdahlii ATCC 55383 and Acetobacterium woodii ATCC 29683 to use their gDNA as template for amplification of Wood-ljungdahl pathway genes.

Cell lysis and Protein precipitation
- Pellet 2 ml of culture by centrifugation, discard the supernatant.
- Add 150 µl TE-buffer and resuspend the cell pellet.
- Add 5 µl of lysozyme (100 mg/ml in ddH2O) and mix by vortexing briefly.
- Incubate at 37 °C for at least 30 min.
- Dilute 1 µl of Proteinase K (50 µg/µl) in 300 µl 2 % SDS solution for each sample.
- Add 300 µl of the Proteinase K/SDS-solution master mix to the sample and vortex briefly.
- Incubate at 65 °C for 15 min, invert every 5 min.
- Cool the samples at 37 °C for 5 min.
- Place the samples on ice for 5 min.
- Add 175 µl of MPC Protein Precipitation Reagent to the sample and vortex vigorously for 10 s.
- Pellet the debris by centrifugation at 4 °C for 10 min at >10.000 g in a centrifuge.
- Avoiding the pelleted debris, transfer the supernatant to a clean microcentrifuge tube.

DNA Precipitation
- Add 1 µl of RNase A (5µg/µl) to the sample and vortex briefly.
- Incubate at 37 °C for 30 min.
- Add 500 µl of ice-cold isopropanol and invert the tubes 40 times.
- Pellet the DNA by centrifugation at 4 °C for 10 min at >1-000 g in a centrifuge.
- Carefully pour off the isopropanol without dislodging the DNA pellet. Centrifuge briefly if the pellet is dislodged.
- Rinse the DNA pellet with 100 µl ice cold ethanol (70 %).
- Remove the ethanol with a pipet tip and dry the DNA pellet at room temperature until the ethanol has evaporated.
- Add 25 µl of EB-buffer (Qiagen) to the dried DNA pellet.
- Place the samples in the fridge and measure next day.

We used gel electrophoresis for verification of PCR amplification and digestion.

- Pour appropriate percentage of Agarose Gel and pour TAE buffer on top
- load 2-3 µl GeneRuler 1 kb DNA Ladder, ready-to-use
- load 5 µl PCR product or digested plasmid + 1 µl 6x Loading Dye
or 2.5 µl digested plasmid + 0.5 µL 6x Loading Dye
- run at 150 V 33 min for PCR verification or at 85 V 50 min for digest verification

We prepped our plasmids out of E. coli and Clostridium saccharoperbutylacetonicum with the Qiagen QIAprep Spin miniPrep Kit according to the manufacturer’s protocol.

To check if our plasmids after transformation have any unwanted mutations, we send them for Sanger Sequencing. The samples for sequencing were prepared as follows:
700 – 1000 ng of plasmid were diluted in to a total of 9 µl and mixed with 4 µl of sequencing primer
Or 400 ng of plasmid were diluted in to a total of 6 µl and mixed with 4 µl of sequencing primer

2x 5 ml LB
250 ml LB in 1 L Erlenmeyer flask with chicane
500 ml sterile ddH2O at 4°C
50 ml sterile glycerol (10% (w/v)) at 4°C
30 sterile labeled Eppendorf cups at – 20°C or -80°C
1x sterile GS3 jar at 4°C
1x Aquatron at 30°C
1x Sorvall RC6 centrifuge at 4°C
1x Universal 320R centrifuge at 4°C
1 L liquid nitrogen

Day 1
- Inoculate 2x 5 ml LB (if required with antibiotics) with your strain of interest, either from cryo-stock culture or from agar plate.
- Incubate overnight at 37°C and 150 rpm.

Day 2
- Inoculate 250 ml LB (without antibiotics) with 2% (5 mL) preculture.
- Incubate in the Aquatron at 30°C and 160 rpm until OD600 of 0.5-0.8.
- Check the culture via microscope for contaminations.
- Let the cells (OD600 of 0.5-0.8) cool down in ice water for 15 min. All further steps are performed under cool conditions.
- Decant the cells in sterile GS3 jar and centrifuge at 4°C and 5000 rpm (4500 g) for 20 min at 4 °C.
- Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 200 ml sterile 4°C ddH2O.
- Centrifuge the GS3 jar at 4°C and 5000 rpm (4500 g) for 5-10 min at 4 °C.
- Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 200 ml sterile 4°C ddH2O.
- Centrifuge the GS3 jar at 4°C and 5000 rpm (4500 g) for 5-10 min at 4 °C.
- Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 30 ml sterile 4°C glycerol (10% (w/v)). Afterwards, transfer the culture into a 50 ml Falcon tube.
- Centrifuge the Falcon tube at 4°C and 6200 rpm for 5-10 min at 4 °C.
- Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 10 mL sterile 4°C glycerol (10% (w/v)).
- Centrifuge the Falcon tube at 4°C and 6200 rpm (4230 g) for 5-10 min at 4 °C.
- Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 500-700 µl sterile 4°C glycerol (10% (w/v)).
- Aliquot the cells into the Eppendorf cups (40 µL per Eppendorf cup). While aliquoting, filled Eppendorf cups must be frozen directly in liquid nitrogen.
- Store the filled Eppendorf cups in a cryobox at -80°C.

2x 5 ml SOB
250 ml SOB in 1 L Erlenmeyer flask with chicane
Ca. 240 sterile labeled Eppendorf cups at – 20°C or -70°C
1x sterile GS3 jar
1x Aquatron at 19°C
1 L liquid nitrogen

Day 1
- Inoculate 2x 5 ml SOB (supplemented with 50 µl 1M MgCl2 and 50 µl MgSO4 and if required with antibiotics) with your strain of interest, either from cryo-stock culture or from agar plate.
- Incubate overnight at 37°C and 150 rpm.

Day 2
- Inoculate 250 ml SOB (supplemented with 2.5 ml 1M MgCl2 and 2.5 ml MgSO4 without antibiotics) with 2% (5 mL) preculture.
- Incubate in the Aquatron at 19°C and 200-250 rpm until OD600 of 0.6.

Day 3
- Let the cells (OD600 of 0.6) cool down on ice for 10 min. All further steps are performed under cool conditions.
- Decant the cells in sterile GS3 jar and centrifuge at 4°C and 5000 U (2500 g) for 10 min at 4 °C.
- Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 80 ml sterile ice-cold TB very carefully.
- Incubation of the suspension for 10 min on ice.
- Decant the cells in sterile GS3 jar and centrifuge at 4°C and 5000 U (2500 g) for 10 min at 4 °C.
- Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 20 ml sterile ice-cold TB very carefully.
- Add 1.4 ml sterile-filtered DMSO.
- Incubation of the suspension for 10 min on ice.
- Aliquot the cells into Eppendorf cups (100 µL per Eppendorf cup). While aliquoting, filled Eppendorf cups must be frozen directly in liquid nitrogen.
- Store the filled Eppendorf cups in a cryobox at -80°C

- Dialysis
- Fill Petri dish ~50 % with sterile demi H2O
- Place Dialysis filter on top of water: shiny side upwards DO NOT TOUCH THE MEMBRANE! USE FORCEPS!
- Carefully place entire reaction mixture on membrane by pipetting. Do not apply to much pressure otherwise it will sink
- Incubate for 30 min at room temperature
- Remove reaction mixture from mebrane by carefully pipetting into new 1.5 mL eppi
- Measure by NanoDrop. Attention! Concentration can be expected to be very low!
- add 2 µL ligation samples to 40 µl defrosted electrocompetent cells
- incubate on ice for 10 min
- transfer cells into precooled electroporation cuvette
- electroporation (2500 V, 25 F, 200 ?)
- directly add 1 mL LB or M9 medium
- incubation for 1h @37 °C
- plating on agar plates supplemented with appropriate antibiotic (here chloramphenicol)

- Thaw 100 µL cell aliquots on ice for 10-15 min
- add 1-5 µL DNA to the competent cells
- incubate cells for 30 min on ice
- heat shock cells @42 °C for 1 min
- place tubes on ice for 5 min
- add 500 µL desired medium to the cells for recovery (M9/LB/SOC)
- incubate cells for 1h @37°C and 210 rpm
- centrifuge cells for 1 min at 13 000 rpm
- discard supernatant and resuspend cells in backflow
- plate all rest on appropriate medium
- incubate plates @37 °C

Preparation:
- Sterile 500 mL anaerobic flask for incubation.
- Channeling into anaerobic tent a few days before: Cuvettes, Eppis, 20 mL syringe, glass beads,
15ml Falcon, channel Falcon centrifuge into the tent.
- Channeling into anaerobic tent at day of transformation: CGM medium, EPB S and EPB NS buffer (with and without MgCl2) and ice-cold metal blocks.
- Epo-machine needs to be connected.

Day1
- Inoculate 1x 5 ml pre-culture with 50 µl cryo-stock culture and incubate at 30°C

Day2
- Main culture is inoculated in an anaerobic flask from pre-culture: 50 mL CGM medium is inoculated to an OD 0.05 at the late afternoon

Day 3
The 50 ml main culture had to be reached an OD 1 - 1.2 (min. OD 0.8, max. OD 1.4).
Competence:
- Culture divided into 4 x 15 mL Falcon (12.5 mL/Falcon).
- Centrifuge set to rcf.
- Centrifuge 2600x g for 12 min.
- Discard supernatant
- Resuspend cells in 5 mL/Falcon (Total 20 mL) in EPB S buffer (with MgCl2).
- Centrifuged again at 2600x g for ca. 12 min.
- Discard supernatant
- Resuspend cells in 250 µL/Falcon (Total 1 mL) EPB NS buffer (without MgCl2).
- Pool the cells in a falcon.

Transformation:
- Add 200 µL of cells into electroporation cuvettes.
- Add 500-1000 ng DNA in 2 mm electroporation cuvettes.
- Incubate cuvettes for 5 min between cold metal blocks before transformation.
- Pulsing: exponential decay wave, 1500 V, 200 Ohm, 25 µF.
- 1 mL CGM was added and gently mixed with the cell suspension.
- Recovery between 2 h or ON at 30°C.
- Cells were shortly centrifuge down, supernatant was discarded and all cells were plated on CGM agar with appropriate antibiotic (here: Thiamphenicol).

Digest of Insert
- Prepare reaction mixture in order indicated:
For one restriction enzyme



For two restriction enzymes



- Incubate time and temperature was chosen according to used enzyme
- Heat shock at 65 °C for 5 min for enzyme deactivation

Digestion of Plasmid
In order to prevent re-ligation of plasmid and Fragment ?? Plasmid is simultaneously cut and dephosphorylated



- Mix thoroughly, spin briefly and incubate at 37 °C for 10 min
- Heat shock at 65 °C for 5 min

Purification of Digest
We purified or digested samples to use for ligation either with the Qiagen QIAquick PCR purification Kit or the Qiagen MinElute PCR
Purification Kit according to their manufacturer’s protocols.
Test digest


- Incubate time and temperature was chosen according to used enzyme
- Heat shock at 65 °C for 5 min for enzyme deactivation

We ligated our desired digested inserts with our different digested plasmid backbones to obtain a plasmid that we later could transform into our solventogenic clostridia.



Ligation was carried out in a 1:5 molar ration of Plasmid to Insert
Formula: required mass insert (g) = desired insert/vector molar ratio x mass of vector (g) x ratio of insert to vector lengths
We used the NEBioCalculator to determine correct amounts.

- Incubate over-night at room-temperature (store in dark)

To store all the strains we obtained during our iGEM period, we prepared cryo-stocks for storage at -80°C. Bacteria strains were grown in appropriate liquid medium. 750 µl of the culture were mixed with 750 µL 99.5 % glycerol. The cryo-stocks were immediately stored on ice until they were transferred to -80 °C.

Gel extraction was performed to specifically purify our 5 kB Insert fragments and to remove the unwanted PCR site-products. We used the QIAquick Gel extraction kit and followed the manufacturer’s protocol.

To test if the plasmids have effects on the growth of our transformed clostridia, growth curves were measured until the stationary phase was reached. Inoculation of 5 ml CGM medium preculture from cryo-stock and incubation overnight at 30 °C. Next day shifting of cultures in fresh 10 ml CGM medium and setting of OD to 0.05. The OD600 for each culture was measured each hour.