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lab notebook: week 1 (6/7 - 6/13)

• Monday 6/7--We successfully defend our project to our sponsors(yay!)
• Wednesday 6/9--First day in lab!--got set up & started cultures of two E. coli strains
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Began growing two strains of E. coli--LE392 chosen as a generally healthy option, and DH5alpha chosen because Keasling et. al. used it in their thiosulfate reductase work.¹

• Spread samples of LE392 (Grindley lab) and DH5alpha (Modis lab) on LB plates and left to incubate overnight at 37
¹ Bang S. W., Clark D. S., Keasling J. D. Cadmium, lead, and zinc removal by expression of thiosulfate reductase gene from Salmonella typhimurium in Escherichia coli. Biotechnol. Lett. 2000; 22: 1331–1335,
• Thursday 6/10--inoculated liquid cultures in AM, made up different copper sulfate & LB solutions, then ran an assay measuring bacterial growth in the presence of copper, trying both strains at three different initial ODs and measuring for four hours
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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

• 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 effect 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
• Friday 6/11--Ran another growth assay with middling concentrations based on results of first, but bacteria failed to grow well--perhaps because allowed to overgrow prior to assay?
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Further Copper Growth Assays—Narrow Concentration Range

Plan to do more assays of bacterial growth in the presence of copper to determine the exact threshold at which growth is affected. Will repeat yesterday’s assay (link) with a more targeted concentration range and a longer growth time.

• Once again inoculate liquid cultures of DH5alpha and LE392 in 5 mL LB using bacteria from plates established on 6/9 (link) and put on shaker to grow at 37˚C
• Let grow for approximately 6 hours, by which point the DH5alpha have reached an OD (measured at 600 nm) of 0.915and the LE392 have reached OD 1.420.
• Diluted them to the three initial ODs chosen on 6/10 (link) in a variety of copper (II) sulfate concentrations chosen to give more detailed data than the wide concentration range trial. The concentrations chosen were 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, 1 mM, 1.25 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 4 mM
• Using these concentrations, ran the copper growth assay overnight using the same protocol as the previous day (link), except that the plate reader took readings for five hours instead of four to better capture all of the growth phase behavior.

These notes can also be found on pages 6-8 of the hard copy lab notebook