Team:Aachen/Notebook/Protocols

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Revision as of 11:28, 10 October 2014

Protocols

2D detection of IPTG and HSL

Chip production

Cell preparation

  1. over night culture of sensor cells (50 mL in a 250 mL flask with) max. 16 h
  2. centrifuge all 50 mL by 3000 g for 10 min at RT (21 °C).
  3. discard the supernatant
  4. re-suspend the pellet in 1 mL tempered (~21 °C) LB-medium .


Agar preparation

  1. autoclave 50 mL medium with 1.5 % (w/v) agarose (has to be multiplied with the number of chips prepared).
  2. cool it down to 45 °C in a water bath.


Chip preparation

  1. mix the cooled medium with the cells by inverting gently.
  2. pour it in the chip form, avoiding bubble formation (!).
  3. wait for approximately 20 min until the agar has solidified.
  4. cut out the chips with a scalpel.
  5. put two chips into a labeled petri dish and store additional 4 chips in labeled petri dishs in the refrigerator.
  6. incubate two chips for 1 h at 37 °C prior to induction.


Aachen 14-10-09 flowsheet chip manufacturingV8 ipo.png
Sensor-chip manufacturing
XXX

Measurement of fluorescence

Culture medium and culture conditions

Media

LB medium

  1. weight components
    5 g/L NaCl
    10 g/L tryptone
    5 g/L yeast extract
    (15 g/L agar for plates)
  2. fill up to 1 L with deionized water
  3. mix well by shaking
  4. autoclave
    1. autoclaving tape, caps slightly unscrewed
    2. base of the pot has to be covered with deionized water
    3. close lid
    4. heat level 3 until the pressure valve opens
    5. reduce heat level to 1.5
    6. set timer to 20 minutes
    7. turn heater off
    8. wait until the pressure valve retracts (30-45 minutes)
    9. open, close caps & shake
  5. for plates, wait until you can touch the bottle (<60 °C, clean bench!)
  6. add antibiotics (1 µL/mL) and shake (gloves!)


TB medium

  1. components 1:
    4 mL/L glycerol
    12 g/L tryptone
    24 g/L yeast extract
  2. fill up to 900 mL with deionized water
  3. mix well by shaking
  4. autoclave
  5. components 2:
    0.17 M KH2PO4
    0.72 M K2HPO4
  6. dissolve in 100 mL deionized water and sterilize it by passing it through a filter
  7. after autoclaving and cooling down, add sterile phosphate solutions


Hartmans minimal medium (HM)

M9 minimal medium (M9)

Components for 1 L Volume
bidest. water 778.667 mL
10x Salt solution 100 mL
Magnesiumsulfatehaptahydrate (10 mM) 100 mL
Glucose 20% (w/v) 20 mL
1000x Trace elements 1 mL
Thiamin (1 mM) 0.333 mL


10x Salt solution
Component' Final concentration Concentration in stock solution
BisTris 95 mM 200,000 mg/L
Ammmonium chloride 60 mM 32,100 mg/L
Sodium citrate 12.5 mM 27,000 mg/L
Monopotassium phosphate 3 mM 4,170 mg/L
Dipotassium phosphate 0.7 mM 1,590 mg/L
1000x Trace elements
ComponentFinal concentration Concentration in stock solution
Iron(III) chloride 50 mM 13,515 mg/L
Calcium chloride 20 mM 2,220 mg/L
Manganese(II) chloride 10 mM 1,258 mg/L
Zinc sulfate 10 mM 1,615 mg/L
Cobalt(II) chloride 2 mM 260 mg/L
Copper(II) chloride 2 mM 269 mg/L
Nickel(II) chloride 2 mM 259 mg/L
Sodium molybdate 2 mM 412 mg/L
Sodium selenite 2 mM 346 mg/L
Boric acid 2 mM 124 mg/L
Hydrochloric acid 1 mM 20 mL


SOC

  1. components
    0,5 % yeast extract
    2 % tryptone
    10 mM NaCl
    2.5 mM KCl
    20 mM MgSO4
  2. fill up with deionized water
  3. adjust to pH 7.5 with NaOH
  4. after autoclaving, add 20 mM sterile glucose solution (filter sterilization)

Genetic methods

Cloning

Restriction Digest

Ligation

Gibson Assembly

The Gibson Assembly was conducted according to the protocol published by New England Biolabs.

  1. Set up the reaction according to the table below on ice (2-3 fragment assembly).
  2. Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.
  3. Transform NEB 5-alpha Competent E. coli cells with 2 μl of the assembly reaction, following the transformation protocol.
Total Amount of Fragments 0.02-0.5 pmols
Gibson Assembly Master Mix (2X) 10 µl
Deionized H2O 10-X µl
Total Volume 20 µl

Transformation

Heat Shock

  1. thaw cells on ice
  2. add 1 µL of plasmid DNA
  3. incubate on ice for 30 min
  4. heat shock at 42 °C for 60 s
  5. incubate on ice for 5 min
  6. add 200 µL of SOC media
  7. incubate at 37 °C for 2 h
  8. plate 20  and 200 µL on plates supplemented with the appropiate antibiotic


Electroporation

  1. add 1 μL plasmid to electrocompetent cells
  2. put DNA/ cell suspension in electroporation cuvette
  3. wipe dry the electroporator
  4. use a small plastic pipette to place the cells
  5. pulse: 2.5 kV, 200-400 Ω, 25 μF (for E.coli)
  6. immediatly add 1 mL LB and incubate for 2 h at 37 °C
  7. plate 50 μL on selective medium plate
  8. centrifuge the rest (3000 g, 20 min), discard supernatant, re-suspend the pellet in 50 μL LB and plate it on selective medium plate

PCR

We have used several different types of PCR throughout our project:

  • colony PCR
  • check PCR
  • gradient PCR
  • SOE PCR
  • touchdown PCR
  • QuikChange(Ligation-During-Amplification)

The scope of appplication as well as the conduct are described below.


Colony PCR /Check PCR

With GoTaq Mast Mix

  • 12.5 µl GoTaq Master Mix
  • 1 µl primer_F
  • 1 µl primer_R
  • pick colony with tip and suspend in PCR tube
  • 9.5 µl ddH2O
parameter duration temp [°C]
denature5:0095
anneal00:3056 30 cycles
elongate01:00 per kb72
denature00:3095
elongate05:0072
storeforever8


gradient PCR

SOE PCR

touchdown PCR

QuikChange

Analytical methods

Agarose gel electrophoresis

Separation of DNA or RNA

  1. take 5µl of the PCR product
  2. mix with 1µl loading dye
  3. apply onto agarose gel together with a marker
  4. run at 120°C for 40 minutes for a full gel

SDS-PAGE

Cell preparation

  • lysis cell pellet in lysis buffer
  • centrifuge for 15 min at 13.000 rpm
  • mix the supernatant with 2x lammli buffer with β-mercaptoethanol
  • denatured for 5 min at 95 °C
  • sample to the gel

For some SDS-PAGEs, we used BioRad ready made gels.

The recipe of the self-made SDS is as follows:

1.5x Buffer

  • 1.5 M Tris-Cl pH = 8.8
  • in 1 L is 40 ml 10 % SDS

Gels

0.75 mm 12 % RUNNING Gel 1 mm 4 % STACKING Gel
1x 2x 4x 1x 2x 4x
H2O 1.65 mL 3.3 mL 6.6 mL 1.5 mL 3 mL 6 mL
1.5x Gel Buffer 1.3 mL 2.6 mL 5.2 mL 0.65 mL 1.3 mL 2.6 mL
30 % Acrylamide (37.5:1) 2 mL 4 mL 8 mL 0.325 mL 0.65 mL 1.3 mL
10 % APS 50 µL 100 µL 200 µL 25 µL 50 µL 100 µL
TEMED 10 µL 20 µL 40 µL 5 µL 10 µL 20 µL

Run gel

  • apply the prepared samples together with a protein marker on the gel
  • run the gel for 10 min at 60 V and after that for ca. 60 min at 120 V

Bradford assay

Determination of protein concentration

Measurement of fluorescence

The measurement of fluorescence was performed using the Synergy Mx (BioTek) microplate reader and the Gen5 software.

  • volume of sample in each well: 100µl
  • measure GFP fluorescence at an excitation wavelength of 496 nm and an emission wavelength at 516 nm

Measurement of optical density

Depending on the number of samples, two different devices were used for measurement of optical density, the Unico Spectrophotometer 1201 (Fisher Bioblock Scientific) and the Synergy Mx (BioTek) microplate reader.