Experiments

Overview:

This page gives the general protocols for several of the experiments we performed this year. If you would like to see the results of these check out our Results and Demonstration page!

Transformation:

Five constructs of plasmid were designed to be utilized as molecular biosensors for phosphate levels within soil. All constructs were synthesized in the medium copy number plasmid “pTwist-kan”. Check out our Parts page to learn more about our constructs!

Electroporation:

  1. Labeled five microcentrifuge tubes with the appropriate plasmid
  2. Transferred 50 µl DH5a electrocompetent E. coli cells to the microcentrifuge tube
  3. Add 5 microliters of the plasmid to the corresponding microcentrifuge tube
  4. Mix gently with the pipet tip
  5. Remove the mixture of cells and ligation reaction from the microcentrifuge tube and add it to an electroporation cuvette making sure all the cells get to the bottom of the cuvette
  6. Place the cuvette in the electroporator, Press and hold the button on the electroporator until a buzz is heard
  7. After the buzz, remove the cuvette from the electroporator and add 1 mL LB broth to the electroporation cuvette quickly!
  8. Remove the solution from the electroporation cuvette and place in a new clean microcentrifuge tube
  9. Pipette the liquid (with cells) from the tube on an LB agar plate containing Kanamycin and spread the liquid on the plate with a plastic spreader
  10. Place the plates in the 37˚ C incubator overnight

Qualitative Color Change:

  • Grew 5 ml liquid cultures overnight of the transformed E. coli strains. As well as two control strains. An E. coli which does not produce any Beta-lactamase as our negative control. An E. coli that produces a lot of Beta-lactamase independent of the phosphate concentration as our positive control.
  • Made five 1:2 dilutions of 0.1 M KH2PO4 buffer
    • Four labeled microcentrifuge tubes had 0.5 ml water added to them
    • (0.1 M) The first (empty) tube had 1ml of the stock concentration of buffer added to it
    • (0.05 M) 500 µl from the first tube was added to the second tube and mixed by pipetting up and down
    • (0.025 M) 500 µl from the second tube was added to the third tube and mixed
    • (0.0125 M) 500 µl from the third was added to the fourth tube and mixed
    • (0.00625 M) 500 µl from the fourth was added to the fifth tube and mixed
  • After the dilutions were prepared:
    • 500 µl of each culture was pipetted in new microcentrifuge tubes labeled the same as the diluted buffers.
    • 125 µl of each buffer dilution was added to each tube.
    • Approximately 5 minutes after the buffer was added, 4 microliters of 10 mg/ml Nitrocefin was added to all tubes
  • Pictures were taken at 5 and 15 minutes after the addition of nitrocefin

Soil Sample Preparation:

We tested two soil samples and two samples that came from biosolids.

  • 2 grams of soil and biosolid samples were added to tubes
  • 10 mM Tris pH 7.5 was added to the samples and vortexed for 30 seconds
  • They were allowed it to incubate at room temperature for 10 minutes
  • E. coli containing our functional construct and nitrocefin were then added to each sample and all samples were allowed to incubate for one hour
  • After one hour, the samples were filtered through a 0.45 micron syringe filter which was attached to a 10 ml syringe and placed in a 15 ml conical tube
  • Absorbance was measured on a spectrophotometer

Spectrophotometry:

To analyze the minute color differences that we can't detect with the naked eye we used two methods of Spectrophotometry.

Light Spectrophotometry:

  • Spectrophotometer ran at wavelength of 510 nm
  • Absorbance readings were taken for the different dilutions described above

UV-Vis Spectrophotometry:

  • Absorbance readings were taken for the different dilutions described above.
  • 1 ml of sample from each tube was transferred to a UV-Vis cuvette
  • 2 mL of DI water was added to bring the sample to an appropriate volume
  • Cuvettes containing these solutions were analyzed by the UV-Vis spectrophotometer at wavelengths from approximately 200 nm to 820 nm
  • Readings from the spectrophotometer were transferred to a spreadsheet file and the data was analyzed