Difference between revisions of "Team:ETH Zurich/Experiments"

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Revision as of 20:15, 19 October 2016

PAVLOV'S COLI

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


Primers were designed according to the guidlines of The Richard Lab 1

  • The targeted mutation should be included into both primers.
  • The mutation can be as close as 4 bases from the 5'-terminus.
  • The mutation should be at least 8 bases from the 3'-terminus.
  • At least eight non-overlapping bases should be introduced at the 3-end of each primer.
  • At least one G or C should be at the end of each primer.
  • Design your primers (including the mutations) to have a Tm >=78°C.

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.

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
Consider the following:
  • 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

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.
        As soon as you out the plate in the plate reader, prepare serial dilutions of DETA/no in PBS.

  • 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
    Note: A separate tube (1 mL) with negative control (no fluorescence) and positive control with very strong fluorescence for calibration (OD = 0.005) also has to be prepared.

References:

  • [1] Tom Richard, Department of Agricultural and Biological Engineering, Penn State University.
  • [2] Gibson, Daniel G., et al. "Enzymatic assembly of DNA molecules up to several hundred kilobases." Nature methods 6.5 (2009): 343-345.

Thanks to the sponsors that supported our project: