Team:ETH Zurich/Experiments
Experimental: Protocols, Methods and Material
Polymerase Chain Reaction - for Construction
New England Biolabs Phusion High-Fidelity DNA Polymerase
Composition:
| Component | 50 µl Reaction |
| Phusion HF Buffer (5x) | 10 µl |
| dNTPs (10 mM) | 1 µl |
| Forward Primer (10 µM) | 2.5 µl |
| Reverse Primer (10 µM) | 2.5 µl |
| Template DNA | variable |
| DMSO (optional) | up to 3% |
| Nuclease-free water | up to 50 µl |
| Phusion Polymerase | 0.5 µl |
Thermocycling Conditions:
| Temperature |
Time |
| 98°C | 30 seconds |
| 98°C 45-72°C 72°C |
5-10 seconds 10-30 seconds 15-30 seconds per kb |
| 72°C | 5-10 minutes |
The appropriate annealing temperature was calculated from NEB's Tm Calculator
Kapa Biosystems Hifi Hotstart Ready Mix
| Component | 50 µl Reaction |
| Master Mix (2x) | 25 µl |
| Forward Primer (10 µM) | 2.5 µl |
| Reverse Primer (10 µM) | 2.5 µl |
| Template DNA | variable |
| Nuclease-free water | up to 50 µl |
Thermocycling Conditions:
| Temperature |
Time |
| 95°C | 3 minutes |
| 98°C 55-75°C 72°C |
20 seconds 15 seconds 15-60 seconds per kb |
| 72°C | 1 minute per kb |
Kapa Hifi Hotstart has similar annealing temperatures as Phusion DNA polymerase, even slightly higher. Only in very few cases a lower annealing temperature was found to be better.
Polymerase Chain Reaction - Colony PCR
For screening a large number of clones, single colonies were resuspended in 20 µl LB medium of which 1 µl was used as template for the PCR.
Solis BioDyne FirePol DNA Polymerase
Composition:
| Component | 8 x 20 µl Reaction (+ 10 µl excess) |
| FIREPol DNA Polymerase | 0.85 µl |
| MgCl2 (25 mM) | 10.2 µl |
| Reaction Buffer B (10x) | 17 µl |
| dNTPs (10 mM) | 3.4 µl |
| Forward Primer (10 µM) | 3.4 µl |
| Reverse Primer (10 µM) | 3.4 µl |
| Template DNA | 1 µl per 20 µl reaction |
| Nuclease-free water | 123.25 µl |
Thermocycling Conditions:
| Temperature |
Time |
| 98°C | 3-5 minutes |
| 95°C 50-72°C 72°C |
30-60 seconds 30-60 seconds 1 minute per kb |
| 72°C | 5-10 minutes |
New England Biolabs Taq DNA Polymerase
Composition:
| Component | 8 x 20 µl Reaction (+ 10 µl excess) |
| Taq Reaction Buffer (10x) | 17 µl |
| dNTPs (10 mM) | 3.4 µl |
| Forward Primer (10 µM) | 3.4 µl |
| Reverse Primer (10 µM) | 3.4 µl |
| Template DNA | 1 µl per 20 µl reaction |
| Taq DNA Polymerase | 0.85 µl |
| Nuclease-free water | 133.45 µl |
Thermocycling Conditions:
| Temperature |
Time |
| 95°C | 5 minutes |
| 95°C 45-68°C 68°C |
30 seconds 20 seconds 1 minute per kb |
| 72°C | 5 minutes |
The appropriate annealing temperature was calculated from NEB's Tm Calculator
Site-Directed Mutagenesis (QuickChange)
| Component | 50 µl Reaction |
| Kapa Hifi Hotstart Master Mix (2x) | 25 µl |
| Forward Primer (10 µM) | 2.5 µl |
| Reverse Primer (10 µM) | 2.5 µl |
| Template DNA | variable |
| Nuclease-free water | up to 50 µl |
Thermocycling Conditions:
| Temperature |
Time |
| 95°C | 3 minutes |
| 98°C 65°C 72°C |
20 seconds 15 seconds 15-60 seconds per kb |
| 72°C | 1 minute per kb |
The resulting PCR product has to be purified (e.x. Agencourt AMPure XP) and digested with DpnI (NEB) for 4 hours and gel-purified. In case Phusion polymerase is used, DpnI can directly be added to the PCR. The product can then immediately be used for transformation. In order to obtain high base exchange efficiency, the template need to be removed from the reaction thoroughly.
Primers were designed according to the guidlines of The Richard Lab 1
Isothermal "Gibson" Assembly2
Recipe for Ready-to-Use Isothermal Assembly Mixes
5x Isothermal Reaction Buffer (6ml)
- 3 ml of 1 M Tris-HCl pH 7.5
- 150 µl of 2 M MgCl2
- 60 µl of 100 mM dGTP
- 60 µl of 100 mM dATP
- 60 µl of 100 mM dTTP
- 60 µl of 100 mM dCTP
- 300 µl of 1 M DTT
- 1.5 g PEG-8000
- 300 µl of 100 mM NAD
This buffer can be aliquoted and stored at -20 °C.
Isothermal Assembly Master Mix
- 320 µl 5x isothermal reaction buffer
- 0.64 µl of 10 U/µl T5 exonuclease
- 20 µl of 2 U/µl Phusion DNA polymerase
- 160 µl of 40 U/µl Taq DNA ligase
- Fill up with water to a final volume of 1.2 ml
The master mix is devided into aliquots of 15 µl and stored at -20 °C.
Protocol for Isothermal Assembly
- 15 µl aliquot of master mix
- 0.02-0.5 pmol DNA in total for 2-3 fragments
- or
- 0.2-1 pmol DNA in total for 4-6 fragments
- Fill up with water to 20 µl
- 2-3 times more insert than backbone (molar ratio)
- 5 times more insert for fragments < 200 bp (molar ratio)
The assembled mix is then incubated for 60 minutes at 50 °C. It can be directly transformed with chemically competent cell (volume of assembly reaction not exceeding 10% of the volume of the competent cells). Otherwise, the reaction mix is purified and desalted (e.x. Agencourt AMPure XP) and a fraction of it transformed with electrocompetent cells.
Preparation of Competent Cells
Preparation of Chemically Competent Cells
- Inoculate 100 ml of prewarmed LB medium with 1 ml overnight culture and grow the bacteria to an OD600 of 0.5.
- Cool the culture on ice, transfer the cells into cetrifugation tubes and harvest them by centrifugation for 5 min (4000xg, 4°C)
- Carfully discard supernatant, keep cells always on ice.
- Resuspend cells in 30 ml cold TFB1 and incubate on ice for 90 minutes.
- Centrifuge for 5 min (4000xg, 4°C)
- Carfully discard supernatant, keep cells always on ice.
- Resuspend the cells in 4 ml ice-cold TFB2 buffer.
- Prepare aliquots of 100 µl in pre-cooled, sterile microcentrifuge tubes and freeze in liquid nitrogen. Store the competent cells at -80° C
Transformation Protocol for Chemically Competent Cells
- Thaw the competent cells on ice
- Use 50 ul of the competent cells for one transformation, do not add more than 5 ul of DNA to the cells (10% of the volume of the competent cells)
- Incubate them for 20 min on ice
- Incubate them for 90 s at 42° C and add afterwards 500 µl of SOC
- Let them regenerate for 1 h at 37° C with shaking
- Plate a part of the culture according to your expectations on LB-agar plates with the approbriate antibiotic.
Preparation of Electrocompetent Cells
- Inoculate 600 ml of prewarmed LB medium with 6 ml overnight culture and grow the bacteria to an OD600 of 0.4.
- Cool the culture on ice fpr 30 min, transfer 300 ml of the culture into 50 ml tubes and centrifuge them for 10 min (3000xg, 4°C). Discard supernatant and add the rest of the culture. Again discard supernatant.
- Carfully resuspend cells in ice-cold, autoclaved and deionized water and fill up to 50 ml
- Centrifuge again for 10 min (3000xg, 4°C) and repeat wash step from above
- Carfully resuspend cells in ice-cold 10% glycerol (working always on ice)
- Centrifuge for 10 min (3000xg, 4°C)
- Discard supernatant
- Resuspend the cells in the remaining glycerol solution.
- Prepare aliquots of 35 µl in pre-cooled, sterile microcentrifuge tubes and freeze in liquid nitrogen. Store the competent cells at -80° C
Transformation Protocol for Electrocompetent Cells
- Thaw the electrocompetent cells on ice. Pre-chill also the electroporation cuvette.
- Add DNA to the cells. Attention: the DNA should contain as little ions as possible. Purify or dilute reactions containing salt.
- Transfer the bacteria/DNA mix into a pre-chilled electroporation cuvette and electroporate them.
- Add IMMEDIATELY 500 µl of SOC and transfer them back into the microcentrifuge tube.
- Let them regenerate for 1 h at 37° C with sufficient shaking.
- Plate a part of the transformation mix according to your expectations on LB-agar plates with the approbriate antibiotic.
Buffer and Medium
TFB1
| Component | Concentration | Amount per 100 ml |
| RbCl | 100 mM | 1.21 g |
| MnCl2 · 4 H2O | 50 mM | 0.99 g |
| Potassium acetate · 4 H2O | 30 mM | 0.29 g |
| CaCl2 · 2 H2O | 10 mM | 0.147 g |
| Glycerol | 15 % | 11.9 g |
Adjust pH to 5.5 and sterilize by filtration
TFB2
| Component | Concentration | Amount per 100 ml |
| MOPS | 100 mM | 0.0047 g |
| RbCl | 50 mM | 0.0027 g |
| CaCl2 · 2 H2O | 75 mM | 0.024 g |
| Glycerol | 15 % | 0.27g |
Adjust pH to 6.5 and sterilize by filtration
LB Medium
| Component | Concentration | Amount per 1l |
| Bacto-tryptone | 1% | 10g |
| Yeast Extract | 0.5% | 5g |
| NaCl | 1% | 10g |
Sterilized by autoclaving
LB Agar
| Component | Concentration | Amount per 1l |
| Bacto-tryptone | 1% | 10g |
| Yeast Extract | 0.5% | 5g |
| NaCl | 1% | 10g |
| Agar-agar | 1.5% | 15g |
Sterilized by autoclaving
M9 Salts 10x
| Component | Concentration | Amount per 250ml |
| Na2PO4· 7 H2O | 128g/L | 31.96g |
| KH2PO4 | 30 g/L | 7.64g |
| NaCl | 5g/L | 1.248g |
| NH4 | 10g/L | 2.495g |
Sterilized by autoclaving
LIV solution
| Component | Concentration | Amount per 10ml |
| L-Leu | 10g/L | 0.1g |
| L-Ile | 3 g/L | 0.03g |
| L-Val | 15g/L | 0.15g |
Sterilized by filtration
Trace elements solution US*
| Component | Concentration | Amount per 50ml |
| FeSO4 | 5.5g/L | 0.25 |
| CaCl2· 2 H2O | 4.1g/L | 0.2g |
| MnCl2· 4 H2O | 1.5g/L | 0.075g |
| ZnSO4 | 1.05g/L | 0.0502g |
| H3BO3 | 0.3g/L | 0.015g |
| Na2[MoSO4]· 2 H2O | 0.25g/L | 0.013g |
| CuCl2· 2 H2O | 0.15g/L | 0.0075g |
| Na2[EDTA]· 2 H2O | 0.84g/L | 0.042g |
Sterilized by filtration
M9* LIV Medium
| Component | Concentration | Volume for 1L |
| M9 Salts | 10x | 100ml |
| Glucose | 50% | 20ml |
| MgSO4 | 1M | 2ml |
| CaCl2 | 0.1M | 1ml |
| Antibiotic | 1000x | 1ml |
| Trace elements solution US* | 1000x | 1ml |
| Thiamine | 10g/L | 2ml |
| LIV Solution | 100x | 10ml |
| H2O | 55.5M | 863ml |
HEPES Buffer
| Component | Concentration | Amount per 50ml |
| NaOH | 5M | 10g |
| HEPES | 100mM | 0.475g |
Adjust pH to 7.0 and sterilize by filtration
Isothermal Reaction Buffer
| Component | Concentration | Amount per 15ml |
| Tris-HCl pH7.5 | 1M | 3ml |
| MgCl2 | 2M | 150ul |
| dNTP | 100mM | 60ul |
| DTT | 1M | 300ul |
| NAD | 100mM | 300ul |
| PEG | N/A | 1.5g |
Sterilized by filtration
Gibson Assembly Mix
| Component | Concentration | Amount per 15ml |
| ITA Buffer | 3.5x | 350ul |
| H2O | 55.5M | 700ul |
| T5 Exonuclease | 10,000units/ml | 0.64ul |
| Phusion DNA Pol | 2,000units/ml | 20ul |
| Taq DNA Ligase | 40,000units/ml | 160ul |
Transferred into PCR tubes in 15ul aliquots
DETA/NO 650mM Solution
| Component | Concentration | Amount per 1ml |
| DETA/NO | N/A | 10mg |
| NaOH | 0.1M | Up to 1ml |
AHL Solution 100mM
| Component | Concentration | Amount per 1ml |
| N-Hexanoyl-L-Homoserine Lactone | N/A | 19.9mg |
| DMSO | N/A | Up to 1ml |
Measurements
Plate reader experiment
-
Day -1: Restreak
Restreak all experiments you want to run from cryostocks
Day 0: Prepare precultures
Prepare precultures of all the experiments you want to run. Prepare 5mL of freshly prepared M9 medium (prepared the same morning), necessary antibiotics, and pick one colony from the plate. Ideally you want to run three biological replicates, but this can be done in three different experiments (the 96-well plate has a finite number of wells and your experiment might not fit). Priority is technical replicates (see setting up the experiment). Keep M9 the in +4 frigde until next day.
Day 1
- Prepare and divide into PCR tubes all PBS amounts necessary to make the dilutions of DETA/NO from the stock. Place PCR tubes in a row next to each other. This eases work with multichannel pipette. Use “experimental calculations” excel sheet to calculate DETA/NO dilutions.
- Open Tecan iController. Open "Heating" on settings and click ON to set temperature to37 degrees before the experiment starts.
OD600 measurement and dilution
- Measure OD600 using the spectrometer. Place 1 mL of liquid (overnight culture) in the cuvette, make sure the arrow on the cuvette aligns with the arrow on the machine. Also don’t touch the lower parts of the cuvette as it can interfere with the measurement.
- Blank spectrometer with M9. If it is not possible to measure OD, dilute the culture 1:1 and try again.
- Dilute preculture to OD=0.1 with M9 and add antibiotics. Take a single packaged sterile 96-well plate. Pipette 200 uL per well and seal completely without touching the seal. Place the plate in the plate reader. Keep in mind the plate reader OD is 1/4th of the actual OD.
- Dilute precultures and let them shake in the plate reader for at least 2 hours before induction to make sure cells are in the exponential phase during induction. Add a blank with M9 media in the well plate.
Setting up the experiment
Design
- Design experiment such that you have triplicates of each sample to be tested.
- On Tecan iController, select the type of plate you are using. The standard one is Greiner 96 Flat Transparent.
- Set the parameters of the Tecan plate reader protocol:
- Temperature: 37 degrees.
- Shaking: 10 seconds, Amplitude 6, Mode: orbital.
- Kinetic Cycle: pick 100 cycles
- Set OD measurement to 600. Set number of flashes to 25.
- add shaking step for 10 seconds with amplitude 6
- add Fluorescence Intensity. For GFP set excitation to 488nm and emission toemission 530nm. The numbers that appear next to nm is the +/- range of the excitation/emission. So pay attention that the range of excitation and emission you get never overlaps, or the measurements will not be accurate. Number of flashes:25. Settle time:0ms. Mode: Top. Z-position: Manual, 20000um. Lag time 0us. Integration time 20us. Label: Green Fluorescence.
- add additional shaking step of 900 seconds with amplitude 6mm and orbital mode and hit start.
Measurements:
- Measurements are collected every 15 minutes. They will be recorded in an excel sheet that automatically opens. It is not recommended to work on tahat excel sheet while the experiment is running.
- Observe OD measurements over time. Always substract it from the M9 blank wells to decide whether or not you have reached the desired OD. You should induce at real OD=0.5 (plate reader OD=0.125). After inducing, put on a new seal. Otherwise condensation interferes with the measurement and also you might cross-contaminate.
Fluorescence-activated cell sorting (FACS)
The protocol was designed by Lukas with the help and consultation of advisers.
- Inoculate 5 mL of overnight culture
- Dilute cells to OD 0.1 in 50 mL flask
- Let cells grow to OD 0.5 and split them into 6 inoculation tubes, with 6 mL of culture per tube
- Induce tubes with appropriate concentrations
- Take samples at different time points:
- Take sample to measure OD and note it
- Take another sample (100 uL): centrifuge it down (4 degrees, 10 000 g, 2 min)
- Resuspend sample in 1 mL 1x PBS
- Dilute sample to OD 0.01 and store it on ice
- Transfer samples on 96-well plates
Kits
Gel recovery
Zymoclean Gel DNA Recovery Kit
The kit is used to recover DNA fragments from agarose gel.
- Supplier: Zymo Research
- Catalog No: D4002
Plasmid extraction and purification
ZR Plasmid MiniprepTM - Classic
The kit is used to isolate plasmid from bacteria.
- Supplier: Zymo Research
- Catalog No: D4016
