Team:NTNU Trondheim/Experiments

Experiments & Protocols

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Alginate solution

  • 3.6 % (w/v) alginate in 0.5 % (w/v) NaCl
  • Stir until completely dissolved
  • Store in the fridge until the next day (to remove most of the air bubbles)

(The type of alginate used during this project is LF10/60 S12727 from FMC Biopolymer, Norway. Properties: FG = 0.65, FM = 0.35, MW = 113500 g/mol, [η] = 635)

Gelling solution

  • 50 mM CaCl2
  • 1 mM BaCl2
  • 0.5 % (w/v) NaCl
  • Autoclave for 20 minutes at 120 °C

Washing solution

  • 2 mM CaCl2
  • 0.5 % (w/v) NaCl
  • Autoclave for 20 minutes at 120 °C

Poly-L-lysine

  • Poly-L-lysine in HBr: 8 mg in 10 ml of NaCl 0.5 % (w/v)
  • Poly-L-lysine in HCl: 10 mg in 10 ml of NaCl 0.5 % (w/v)

Washing solution (for poly-L-lysine-coated capsules)

  • 300 mM mannitol in sterile water

LB medium for capsule leakage tests

  • 2mM CaCl2 in standard LB + 35 μg/ml Kanamycin

PBS medium for capsule leakage tests

  • PBS 1X + 35 μg/ml Kanamycin

m-Toluic acid (inducer for P. putida pHH+GFP)

  • 1 M in EtOH (working concentration 1 mM)

Preparation of cell culture

Overnight inoculation of P. putida

  • Inoculate P. putida from -80 oC freezer in 3 ml LB + 35 μg/ml Kanamycin
  • Incubate overnight at 30 oC

Equipment needed for generating alginate microcapsules

  • Electrostatic capsule generator (with a needle suitable for generating capsules of desired size)
  • Syringe feeder and disposable sterile syringes
  • Surgical tubing for connecting the needle to the syringe
  • Magnetic stirrer
  • Glassware
NOTE: Apart from the electrostatic capsule generator and the syringe feeder, all equipment and glassware should be sterilised prior to use.

Preparation of alginate/cell mixture

  1. Centrifuge ON culture and pour off supernatant. Resuspend cells in PBS 1X.
  2. Adjust OD to 0.1.
  3. Carefully mix the alginate solution and cell culture 1:1 (the concentration of alginate in the mixture should be 1.8 % (w/v)), without incorporating air to the mixture.
  4. Place the desired volume (0.5 - 3.0 ml) into a disposable 5 ml syringe (BD Plastipak)

Cell encapsulation

  1. Connect the syringe containing the alginate/cell mixture to the needle by surgical tubing.
  2. Place the syringe in the syringe feeder, find the correct syringe type and dimensions in the instrument’s menu. Set syringe feeder speed.
  3. Place the syringe feeder close to the electrostatic capsule generator, and place the needle in the needle holder.
  4. Pour gelling solution in a short and wide glass containing a magnet, and start the magnetic stirrer underneath.
  5. Adjust the needle holder’s distance to the gelling bath surface.
  6. Set a suitable voltage on the electrostatic capsule generator, and flick the start-switch.
  7. Wait until all of the alginate/cell mixture has passed through the needle.

(For generating capsules with a diameter of ~230 μm the following parameters were chosen: inner diameter of needle 130 μm, speed 6 ml/hour, voltage 5000 V, distance between needle and gelling bath 1 - 2 cm)

Capsule wash

  1. Let the capsules stay in the gelling bath for a few minutes.
  2. Gently pour the gelling solution containing the capsules through a filter (mesh, pore size 40 μm), while holding the filter holder tube at an angle.
  3. Put the filter holder containing the capsules in an unused and sterile beaker, gently flush the capsules with the washing solution (mannitol solution if coating the capsules in poly-L-lysine) using a 25 ml graduated pipette (repeat 2 times).
  4. Put the filter holder containing the capsules in an unused and sterile beaker, gently resuspend the capsules in the desired volume of storage medium or continue with coating the capsules in poly-L-lysine.

Poly-L-lysine coating of capsules

  1. Prepare fresh poly-L-lysine solution.
  2. Following the cell encapsulation, wash the capsules in mannitol (as described under the capsule wash protocol).
  3. Transfer the capsules to a poly-L-lysine bath (with magnetic stirrer).
  4. Wait 10 minutes before filtering the capsules (as described under the capsule wash protocol).
  5. Gently pour the poly-L-lysine solution containing the capsules through a filter (mesh, pore size 40 μm), while holding the filter holder tube at an angle.
  6. Put the filter holder containing the capsules in an unused and sterile beaker, gently flush the capsules with 0.5 % (w/v) NaCl solution using a 25 mL graduated pipette (repeat 2 times).
  7. Put the filter holder containing the capsules in an unused and sterile beaker, gently resuspend the capsules in the desired volume of storage medium.

Preparation of samples

  1. Overnight inoculation at 37 oC of each biological replicate of devices 1-3 + positive and negative controls in 3 ml LB + Kanamycin (Chloramphenicol for the controls).
  2. 5 % inoculation at 37 oC in 3 ml LB + Kanamycin (Chloramphenicol for the controls).
  3. Measure OD, and dilute/adjust OD to 0.3 (+/- 0.025). This was found (by trial and error) to be a suitable density of cells for confocal microscopy.
  4. Cell cultures were then put on ice and transported to the microscope.
  5. For each biological replicate, 1.5 μl of the sample was put on a rectangular cover glass of thickness 1.5 (served as a suitable object glass for confocal microscopy). A small square-shaped cover glass of thickness 1.5 was put on top of the sample. To reduce bacteria moving and being pulled towards the edges of the cover glass, its edges were sealed with nail polish.
  6. For each prepared microscopy sample (biological replicate), three images were obtained to serve as technical replicates. These three images should be taken at different xy positions in the prepared microscopy sample.

NOTE: The small volume of 1.5 μl ensures a short enough distance between the glasses (to work well with the z section size set by the pinhole size, see below), so that all bacteria between the glasses in a given xy(z) frame can be in focus and thus their fluorescence be detected by the microscope.

Acquisition of images by confocal microscopy

  1. A Leica SP5 confocal microscope was used with the following parameters set in the LAS AF software: Argon laser (power 20 %) excitation line 488 nm (set to 6 %), hybrid detector (HyD, Leica) set to filter and detect emitted light between 495-600 nm, pinhole size 399.99 nm (with a corresponding z section thickness of 2.897 μm).
  2. The objective used was HCX PL APO CS (63x magnification, water immersion, numerical aperture 1.2, XY resolution 163 and Z resolution 290 at 488 nm).
  3. Look into the microscope to focus in on your bacteria (bright field).
  4. For live view, set the format to 512 * 512 and speed to 400 Hz, and press ‘Live’.
  5. Adjust the focus with the z-position wheel, and when a good view of the bacteria and their fluorescence has been found in live view, stop the live view to scan the image. The following parameters were set in the LAS AF software to acquire the images: bit-depth of 12 (found under the configuration tab), acquisition mode xyz, zoom 1.25, speed 100 Hz, format 2048 * 2048 (pixel size 96.15 nm * 96.15 nm, image size 196.83 μm * 196.83 μm), line average 3.
  6. The Leica SP5 should be set to acquire both a fluorescence image (with the HyD detector) and a regular bright field image (it is important to set both channels to ‘Visible’). The settings for the bright field channel were Smart Gain 305 V, Smart Offset -2.5 %. The setting for the fluorescence channel was Smart Gain 10.0 % (the HyD detector sets the offset by itself).
  7. Each captured image (containing TIFF files of both bright field and fluorescence images) should be saved automatically under the same experiment (LIF file). This enables the microscopist to export the LIF file, which is then ready for the subsequent analysis.

NOTE: A bit-depth of 12 was chosen in this Interlab study to ensure that the range would accommodate both the samples with strong fluorescence and weak fluorescence. A lower bit-depth like 8 bit would cause the images with strong fluorescence to be oversaturated because the range would simply not be wide enough (data lost before analysis).

Quantitative image analysis

  1. The data set was provided by the microscope software in LIF format. This file has about 1GB size. While ImageJ can read LIF format, it does not manage the memory very well, and cannot load the images simultaneously. The method used to circumvent this issue is to convert the LIF file into many TIFF files. Each TIFF file contained one fluorescence image and one bright field image. bfconvert utility was downloaded to convert the Leica LIF files into a set of TIFF files. The utility was downloaded from http://downloads.openmicroscopy.org/bio-formats/5.1.3/.
  2. The images were converted with the command line: 
    $ ./bfconvert 150818\ Interlab\ Hyd2.lif %n.tiff
  3. The resulting files have spaces in their name. This created problems for ImageJ. The spaces were converted into underscores with the following command: 
    $ for file in *.tiff; do mv "$file" `echo $file | tr ' ' '_'` ; done
  4. The ImageJ analysis was scripted into a macro file. The commands of the macro file are as described in the following instructions.
  5. The TIFF image was opened with ImageJ.
  6. The two stacks of the images were converted into two images for easy reference in the macro.
  7. The bright field image was selected and viewed with a Grays LUT.
  8. The background was subtracted using Process > Subtract background...". In the new window, the rolling ball radius is 50 px, and the "Light" was ticked. A new window appeared asking whether to process the images. "No" was clicked.
  9. The threshold was determined by clicking Image > Adjust > Threshold… "Apply" was clicked. On the new window, Background: Light was selected. Calculate threshold for each image was unticked. "Apply" was clicked/.
  10. Analyze > Set Measurements was selected, "Area", and "Mean" are ticked, and Redirect to: was set the name of the first image (fluorescence).
  11. Analyze > Analyze Particles was clicked. For size, 2E-8 was set as the lower bound. For circularity, 0.10 to 0.7 was set, and show outline was selected for visualization purposes.
  12. Results window was selected, and saved as a CSV file.
  13. In MATLAB, all the batch processed files were converted into a 3D matrix TABLE (Devices, Biological replicates, Technical replicates), the intensity was calculated as MEAN(TABLE,3), which is the average over the technical replicates. These results were satisfactorily compared with the flow cytometry results.

LB medium

  • 10 % Tryptone
  • 5 % Yeast extract
  • 5 % NaCl
  1. Autoclave for 20 minutes at 120 °C
  2. Add appropriate antibiotics up to respective working concentration

LA medium

  • 10 % Tryptone
  • 5 % Yeast extract
  • 5 % NaCl
  • 20 % Agar
  • Autoclave for 20 minutes at 120 °C
  • Add appropriate antibiotics up to respective working concentration

Antibiotic stocks (according to iGEM protocol)

  • Ampicillin: 100 mg/ml in 50 % EtOH
  • Chloramphenicol: 35 mg/ml in 100 % EtOH
  • Kanamycin: 35 mg/ml in distilled H20
  • Aliquots of 500 μl, stored at -20 °C

Making P. putida electrocompetent

  1. Inoculate of 1 % from ON culture in 10 ml of LB medium
  2. Incubate at 30 °C until OD600 = 0.45 +/- 10 %
  3. Aliquot 1.5 ml of cell suspension in an eppendorf tube per electroporation
  4. Centrifugate at 12000 G for 1 minute at 4 °C
  5. Remove supernatant
  6. Add 1 ml 300 mM sucrose (cold)
  7. Centrifugate at 12000 G for 1 minute at 4 °C
  8. Remove supernatant
  9. Add 50 μl 300 mM sucrose (cold)
  10. Keep on ice until used for electroporation

Electroporation

  1. Add plasmid (1 - 5 μl, up to 500 ng DNA) to 50 μl electrocompetent cells into electroporation
  2. Electroporation with Biorad GenePulser Xcell
    • Voltage: 2500 V
    • Capacitance: 25 uF
    • Resistence: 200 Ohm
    • Cuvette: 2 mm
  3. Add 1 ml LB medium to the electroporation chamber immediately after pulse
  4. Transfer all liquid from the electroporation chamber into an eppendorf tube
  5. Incubate for 1 hour at 30 °C
  6. Plate cells

Plating

  1. Transfer 50 μl of cell suspension in the middle of the LA plate (with appropriate antibiotic)
  2. Distribute cells equally over the whole plate
  3. Centrifuge remaining cell suspension and remove supernatant until less then 100 μl remains
  4. Resuspend cells in remaining supernatant
  5. Transfer all remaining cell suspension onto a second LA plate
  6. Distribute cells equally over the whole plate

Heat transformation of E. coli

  1. Add plasmid (1 - 5 μl, up to 500 ng DNA) to 100 μl competent cells (stored at -80 °C) in eppendorf tube
  2. Keep on ice for 30 minutes
  3. Keep in water bath for 45 seconds at 42 °C
  4. Keep on ice for 3 minutes
  5. Add 500 μl LB medium
  6. Incubate for 1 hour at 37°C

Miniprep: Preparation of DNA from E.coli

According to Wizard Plus SV Minipreps DNA Purification system

Nanodrop: Measurement of DNA concentration

Enzyme digest (according to iGEM protocol)

  1. Keep all enzymes and buffers used on ice
  2. Add 250 ng of DNA to 3 μl of Cutsmart Buffer and 0.5 μl of each appropriate enzyme. Fill up with nuclease free water to a total volume of 16 μl
  3. Incubate mixture for 30 minutes at 37 °C (enzyme digest)
  4. Incubate mixture for 20 minutes at 80 °C (heat inactivation)

Ligation (according to iGEM protocol)

  1. Add 25 ng of DNA to 1 μl T4 DNA Ligase buffer and 0.5 μl T4 DNA Ligase. Fill up with nuclease free water to a total colume of 10 μl
  2. Incubate for 30 minutes at 16 °C (ligation) *or overnight
  3. Incubate for 20 minutes at 65 °C (heat inactivation)
  4. Transformation with 2 μl of product

Polymerase Chain Reaction (PCR)

Recipe
  • 2.5 μl forward primer
  • 2.5 μl reverse primer
  • 1 μl dNTP
  • <1000 ng template DNA
  • 0.5 μl Q5 polymerase
  • 10 μl Q5 buffer
  • 10 μl High GC enhancer
  • Fill up with nuclease free water to a total volume of 50 μl
Protocol
  1. 30 seconds - 98 °C
  2. 10 seconds - 98 °C
  3. 20 seconds - Annealing temperature
  4. 80 seconds - 72 °C
  5. Repeat steps 2.-4. 20-30 times
  6. 2 minutes - 72 °C
  7. Infinite hold at 4 °C

Gelelectrophoresis

Spectrophotometry

Gel extraction

Flow Cytometry

  1. Inoculate 1 % of overnight culture in 3 ml LB medium
  2. Incubate at the appropriate temperature (30 °C for P.putida, 37 °C for E.coli)
  3. Dilute 1:100 in PBS, total volume: 1.5 ml
  4. Transfer 500 μl in flow cytometry tube (per technical replicate)
  5. Flow cytometry with BD Accuri C6 sampler
    • Suction: 2 minutes
    • Flow rate: 35 μl/minute
    • Pre-set thresholds: FSC = 4000, FL-1 = 200
    • Washing steps after last technical replicate from each sample

PBS

HIFI

TE Buffer

Retrieved from "https://2013.igem.org/Team:Cornell/notebook" and "https://2014.igem.org/Team:NTNU_Trondheim/notebook"