IGEM:Yale/2010/Notebook/2010/06/10

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Baseline Wide Concentration Range Copper Growth Assay

Set out to find baseline growth exhibited by the E. coli strains being used when grown in a variety of copper concentrations. This will serve as a basis for comparison once copper tolerance has been engineered. Chose a range from the very, very low (sure to have no effect) to the very, very high (surely lethal).

Set-up

  • Studies were conducted in a 96-well plate, and growth was measured with a plate reader.
  • 12 concentrations of copper(II) sulfate/LB solution were used with final (post-E. coli addition) copper(II) sulfate concentrations of 1 M, 0.1 M, 50 mM, 10 mM, 1 mM, 500 uM, 250 uM, 100 uM, 10 uM, 1 uM, 0.1 uM, and 0 uM, and each concentration was given its own column in the well plate. A row of cells with just the copper solutions was included to function as blanks.
layout of 96-well plate
layout of 96-well plate
  • Each of the two E. coli strains (LE392 and DH5alpha) was grown with each of the copper concentrations from three different starting densities. These three initial densities (OD 0.075,OD 0.0375, and OD 0.01875) were chosen so that mid-log phase would be reached at 1.5 hr, 2 hr. and 2.5 hr respectively after the start of the trial.
  • Absorption measurements were taken approximately every 10 minutes for four hours. Lack of a 600 nm filter necessitated measurement at 540 nm, a wavelength at which copper (II) sulfate absorbs, but the use of the appropriate blanks prevented this from interfering with data collection.
  • Samples were kept at 37 with alternated minutes of orbital shaking and stillness (plate reader does not allow continual shaking).

Results & Conclusions

  • Both cells lines exhibit very similar copper tolerances, growing without noticeable affect at concentrations up to 500 uM, experiencing slowed growth at 1 mM, and no growth at 10 mM.
  • More trials are necessary at concentration ranges between 500 uM and 10 mM to determine more exactly the maximum copper tolerance of these cell lines.
  • After four hours of growth the healthier cells were still growing very rapidly. Future assays will extend recording time to five hours in an effort to capture the plateau region.

This work is also documented on pages 1-5 in the hard copy of the lab notebook

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