Recipes for construction
| Primer 1 | 50 μL |
| Primer 2 | 50 μL |
| dNTP | 50 μL |
| Buffer | 100 μL |
| MgCl2 | 100 μL |
| dH2O | 600 μL |
PCR mixture
| Component | For 1 reaction (V=10µl) | For 1000 reactionsL |
| Forward primer | 0,5 μL | 50 μL |
| Reverse primer | 0,5 μL | 50 μL |
| Master mix k0171 | 5 µl | 500 μL |
| dH2O | 4 μL | 400 μL |
Restriction mixture
| DNA | 20 μL |
| buffer Tango | 2,5 μL |
| restriction enzyme 1 | 1 μL |
| Restriction enzyme 2 | 1 μL |
Ligation mixture
| Component | Volume (µl) |
| insert | 6 μL |
| vector | 2 μL |
| T4 DNA ligase | 1 μL |
| T4 DNA ligase buffer | 1 μL |
Incubate over night at 16°C or 2 hours at room temperature.
Ligation mixture for pJET blunt vector
| Gene | 7 µl |
| Vector | 1 μL |
| T4 DNA ligase buffer | 1 μL |
| T4 DNA ligase | 1 μL |
SDS-PAGE gel (12,5 %)
| Component | Separation gel (V= 10ml) | Stacking gel (V= 5ml) |
| dH2O | 4, 27 ml (5x854 µL) | 3, 08 ml (4x769 µL) |
| separating gel buffer | 2, 5 ml (3x833 µL) | / |
| stacking gel buffer (pH= 6,8) | / | 1, 25 ml (2x625 µL) |
| acrylamide bisacrylamide (37:1) | 3,13 ml (4x783 µL) | 0, 625 ml (625 µL) |
| 10% SDS | 100 µL | 50 µL |
| TEMED | 15 µL | 7,5 μL |
| 15 % APS | 30 μL | 15 μL |
| Component | For 1 litre | for 500 ml |
| Bacto-tryptone | 9 g | 4,5 g |
| Yeast Extract | 4,5 g | 2,25 g |
| Sodium chloride | 4,5 g | 2,25 g |
• Broth should be autoclaved (sterilization)
• Broth should be stored at room temparature until opened and then must be stored at 4°C
LB agar for bacterial cell culture plates
Mixture:
| Component | For 1 litre | for 500 ml |
| Bacto-tryptone | 9 g | 4,5 g |
| Yeast Extract | 4,5 g | 2,25 g |
| Sodium chloride | 4,5 g | 2,25 g |
| Bacto Agar | 1,5 g | 0,75 g |
• Once autoclaved the LB AGAR can be used as soon at it has cooled to ~55oC, or it may be stored at room temperature and microwaved when required
Colouring fluid
• 20% acetic acid and Comassie Brilliant Blue dye must be mixed 1:1 (20µL:20µL).
Fluid for discoloration
• 30% ethanol, 10% acetic acid
Comassie Brlliant Blue
• Mix 1 g of comassie Brilliant Blue powder with 200 ml ethanol 300 ml H2O
Separating Buffer
• 1,5M Tris/HCl, pH= 8,8
Stacking Buffer
• 0,5 M Tris/HCl, pH= 6,8
10xSDS Buffer
• 30g Tris, 144g Glycine, 10 g SDS (sodium dodecyl sulfide)
Preparation of western transfer buffer
| Tris | 3 g |
| Glycine | 14,4 g |
| Methanol | 200 μL |
| H2O | 800 μL |
| NaCl | 5,8 g |
| Na2HPO4 | 8,9 g |
Binding buffer
•20mM Hepes
•500 mM NaCl
•20mM imidazole
•fill with H2O till 250 ml
•pH=7, 5
Elution buffer
•20mM hepes
•500mM naCl
•250 mM imidazole
•fill with H2O till 250 ml
•pH=7,5
Recipes for testing
Bioreactor start-upFirstly, 2 grams of glucose are added into a bench-scale batch bioreactor (full volume of 650 mL, working volume of 400 mL), to provide sufficient energy source for the mixed anaerobic microbial culture, present in the inoculum. In the beaker, 300 g of inoculum and 100 g of deionized water are precisely weighted for each bioreactor, to allow for a simultaneous preparation of 12 bioreactors. At that point, pH of the solution in each bioreactor is adjusted to 5.7 by means of 2 molar hydrogen chloride. When setting the appropriate pH, the solution is intensely mixed in order to obtain a homogenous solution. The prepared solution is then transferred to each bioreactor (400 g) and the liquid content is purged with Argon for 10 minutes. The reactor contents are sampled and frozen at -20 °C until further analysis. After sampling, the bioreactors are sealed and the integrated mixing devices are activated (200 RPM). The bioreactor is fitted with two sampling ports (one for liquid sampling - we analysed these samples later on a HPLC device, the other for online analysis of the produced gas by GC apparatus) and connected to the gas-measuring flow-through cells. After full assembly of the bioreactors, the headspace (gas space above the liquid broth) of the bioreactor is purged, for 5 minutes. Argon is used because it ensures anaerobic environment, is not produced during our fermentation process (unlike e.g. N2) and does not form compounds with the substances in the reactor or represent a source of food for the inoculum (unlike e.g. CO2). Simultaneously with the test-run, two bioreactors are incubated without any added glucose. These 'blind’ runs serve as a control of inoculum activity.
Preparation of samples for HLPC measurementsApproximately 2 ml of bioreactor contents is sampled through an integrated sampling valve. The precise volume is recorded and exchanged with the same amount of argon, to maintain anaerobic conditions. The samples are marked, weighted and centrifuged (10000 RPM, 20 min). The supernatant is then diluted with ultrapure water and filtered through a 22 µm syringe filter into marked HPLC vials, which are then transferred to HPLC auto sampler for analysis.
HPLC analysis (Agilent 1260 Infinity HPLC, Agilent Technologies)Prior analysis, the system is equilibrated by performing 3 injections / vial from 2 vials of fresh ultrapure water. HPLC set-up and conditions:
• HI-Plex colon (Agilent Technologies, 7.7x 300mm, 8 µm)
• DAD detector (butyric acid was analyzed at 210 nm wavelength).
• Colon temperature: 50 °C
• Mobile phase: 0.01 M H2SO4
• Flow: 0.6 ml / min
• Injection volume: 20 µ
l
Preparation of standard ENDO growth medium
For 100 ml of ENDO medium (BUJON) with lactose and antibiotic combine:
• 1,00 g of meat peptone
• 1,00 g of lactose
• 0,25 g of sodium sulfite
• 0,35 g of dipotassium hydrogen phosphate
• Add ultrapure water to total volume of 100 ml.
Preparation of peptone water (250 ml) - minimal growth medium
• 1,50 g of peptone
• 1,75 g of sodium chloride
• Add ultrapure water to total volume of 250 ml.
General instructions for media preparation
Prepare a solution of HCl and solution of NaOH in advance. These solutions serve as the pH regulators.
HCl solution: dilute 20 ml of concentrated (37%) HCl in 100 ml of ultrapure water.
NaOH solution: carefully dissolve 40 g of NaOH in 200 ml of distilled water.
Use protective glasses and be careful; the solution can boil!
Media G1-G4 prepare according to the composition table using Falcon (50 ml) centrifuge tubes.
Butyric acid (ρ = 0,95 g/mL) is measured with pipette:
• 0,12 g = 126 µL
• 0,080 g = 84 µL
• 0,040 g = 42 µL
Glycerol, lactose, NaCl and yeast extract are weighed.
Other media are prepared in 2000 ml flasks using following chemicals:
• 20 g of meat peptone
• 7 g of dipotassium hydrogen phosphate
• 5 g of sodium sulfite
All chemicals are weighted with analytic balance in the weighing trays, which must be pre-cleaned and thoroughly dried prior weighting. Add a few milliliters of ultrapure water to dissolve the weighted chemicals and then fill the flask to 95 vol. %. Adjust pH and fill the flask to the mark. First prepare the flask with pH=5, then repeat the procedure for pH = 6, pH=7, pH=8.
Media G5-G68 are prepared following these instructions:• Put the beaker with the empty Falcon (50 ml) centrifuge tube on the balance and press TARE.
• Weigh 35 g of solution from the flask and pour the chemicals in it. Always wash the weighing boats with water and pour the water in the Falcon centrifuge tube.
• Add 39 g of media from the flask and adjust the pH value accordingly with appropriate NaOH or HCl solution.
• Place the tube on the Vortex machine for 20 seconds to achieve good dissolution of the compounds.
• Check the pH value again and if not adequate, adjust again!
• Fill the centrifugate tube with ultrapure water up to 40,00 g and close it.
Inoculation of genetically modified E.coli
Bacteria are inoculated on freshly prepared LB plates in a laminar:
• One colony is taken from the previous plate with an inoculation loop.
• Gently drive the loop over the surface of a fresh plate (6-times left and right, one down and another left to right).
• Cover the plate and wrap with parafilm.
• Mark well (date, type of bacteria, sample, medium).
• Incubate at 37°C.
Instructions for an autoclave
Fill the autoclave and make sure, the glassware and other contents is not tightly packed. Close the autoclave and tighten firmly. Check the level of water and add fresh water when necessary. Then, close the steam valve and turn on the thermostat. Autoclave 20 minutes at 1.21 bar internal pressure. Leave to cool before opening the lid.
Preparation of physiological solution (0,9 wt. % NaCl )Dissolve 4,5 g of NaCl in 500 ml of ultrapure water. Do not autoclave more than once.
Sterilization of antibiotic (e.g. removal of resistant bacteria)
Aseptically filter the antibiotic solution through a 22 µm syringe filter into a sterile micro centrifuge tube.
Biosafety cabinet
Activate and set the appropriate cabinet conditions well in advance (approx. 10 minutes prior use) and wait for the chamber flow and filter system to stabilize. Work in the direction from the sterile to the infected side of the chamber and never in the opposite direction! When you finish, disinfect all objects before you remove them from the biosafety cabinet. Turn off the biosafety cabinet.
Analysis of waste glycerol• CHNS analysis
• Karl-Fitcher titration
The sample is diluted with water by the factor 100 and filtered through the filter into a vial. Then, standard HPLC analysis is done.
Pipetting robot connection settings
• Change adapter-LAN-properties-PV4+ code (IP address)
IP addresses:
192.168.65.67
192.168.65.14
255.255.255.0
193.2.1.66
192.168.65.254
• Second icon: ‘PING’ + ‘address + 137’ → ‘send 4, receive 4’
The method of butan-1-ol isolation
• Ultrapure bio-filtered water is used (18,2 Ω/cm)
• 5 wt. % solution of butan-1-ol is prepared
• 10 ml is transferred in the 100 ml flask and diluted to achieve 0.5% solution
• 10 ml is transferred in the second 100 ml flask and diluted to give 0.05% solution
• 1 wt. % solution of butan-1-ol is prepared
• 1.1 ml of solution is taken out of each flask and divided amongst 3 microcentrifuge tubes
• Then, place the tubes into a centrifuge and spin:
1. at 5000 rpm for 1 minute
2. at 5000 rpm for 5 minutes
3. at 5000 rpm for 10 minutes
4. at 10000 rpm for 1 minute
5. at 10000 rpm for 5 minutes
6. at 10000 rpm for 10 minutes
Before the centrifugation process prepare:
• 100 ml of 0.25 M potassium dichromate
• 100 ml of 0.1 M silver nitrate
• 100 ml of 6 M sulphur (VI) acid
• After that, prepare 4 centrifuge tubes following the next protocol:
o 5 ml of potassium dichromate solution in each
o 1 drop of silver nitrate
o Mix immediately!
o Add 5 ml of H2SO4
o Mix again
o
The centrifugation process:
• 100 µl is transferred from the microcetrifuge tubes to new microcetrifuge tubes and 900 µl of ultrapure water is added,
• 5 ml of potassium dichromate solution in each,
• 1 drop of silver nitrate,
• mix immediately!
• Add 5 ml of H2SO4 and
• mix again.
The standardized protocol is repeated for all cycles, all measurements are repeated three times (18 cycles).
Preparation of photobacterium agar
• Casein enzymatic hydrolysate 5.0 g/L
• Yeast extract 2.5 g/L
• Sodium chloride 30.0 g/L
• Ammonium chloride 0.3 g/L
• Magnesium sulfate 0.3 g/L
• Ferric chloride 0.01 g/L
• Calcium carbonate 1.0 g/L
• Monopotassium dihydrogen phosphate 3.0 g/L
• Sodium glycerophosphate
Add ultrapure water to achieve total volume of 250 ml.
We additionally add 15 g/L of agar. The solution is heated and autoclaved. Then, it is spilled over the petri dishes (0,3 cm of height) and left to be cooled down.
Preparation of Escherichia coli biofilm
ENDO medium (BUJON) with lactose and antibiotic:
• 1.00 g of meat peptone
• 1.00 g of lactose
• 0.25 g of sodium sulfite
• 0.35 g of dipotassium hydrogen phosphate
Add ultrapure water to achieve 100 ml total volume.
Peptone water:
• 1.50 g of peptone
• 1.75 g of sodium chloride
Add ultrapure water to achieve 250 ml total volume.
Add ultrapure water to the labelled mark in both solutions. Both media are placed in the autoclave simultaneously with the holders for 50ml biofilm. When all items are sterilized, they are placed directly in the laminar. At the moment when the temperature of the both media is 60°C sterile antibiotic is added (1 µl/ml of medium). After the ENDO medium reaches room temperature, the bacteria is transferred from LBC medium. The conical flask then incubated on the shaker (100 RPM) for 12 hours at 37°C and is used for the transmission of the medium into sterilized 26-well plate the next day. In each well 100 was added with 100 µl of diluted solution of bacteria in peptone water, diluted in the ratio 1:100. Tega Stavka ne razumem. During the following incubation at 37 °C the inoculated growth medium was kept still.