Selecting for Tetracycline Sensitive E. coli

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Reasons For Selection:

Making selective media is beneficial for multiple reasons. With these specific plates, we can select for strains of bacteria with a riboswitch connected to a tetracycline fitness module that are "off" when they are supposed not exposed to their ligand. This could allow for the screening of multiple mutations of riboswitches, and all us to choose viable options to continue working with. It is important that the riboswitch is not "leaky" when not exposed to the ligand, because a leaky switch would allow for survival of those cells which are not actually exhibiting programmed evolution.

Although not tested yet, these plates should allow for introduction of ligand. This would allow experimenters to spot different strains that have already been shown to have an "off" riboswitch, to see if they are still "off" when the ligand is present. Those strains could then be thrown out as options for further testing.

Plate Ingredients:

  • Agar - 15 grams
  • Tryptone - 4 grams
  • Yeast Extract - 4 grams

Autoclave for 30 minutes at 121 C

  • Fusaric Acid (2mg/mL) - 6mL
  • ZnCL (20mM) - 5mL
  • Ampicillin (100mg/mL) - 200uL
  • NaH2PO4 (100mg/mL) - 100mL

(Amounts in 1L of Water)

The first plates were originally made from the ingredients found in the papers from Bochner, B.R. et a[[1]]l, and Maloy, S.R, and W D Nunn[[2]].

After finding that these plates did not select as expected, we made a few changes to the ingredients that allowed for growth at 100x dilution of tetS (tetracycline sensitive) cells, but not tetR (tetracycline resistant cells).

Procedure for spotting bacteria:

  1. Grow separate overnight cultures of the cells
  2. Dilute each culture 100 fold (99uL of water for every 1 ul of culture) to an OD of about ___
  3. Plate approximately 2uL of the dilution on the selective plates
  4. Spots that grow are tetracycline sensitive cells, spots that don't grow are the tetracycline resistant cells(those with leaky riboswitches)

Procedure for spreading bacteria:

  1. Grow overnight cultures of the cells (can be many different strains grown together)
  2. Dilute each culture 100 fold (99uL of water for every 1 ul of culture) to an OD of about ___
  3. If the strains are not already mixed, you can mix the different dilutions in 1:1 ratios
  4. Take 1uL of the mixed dilutions and add to __water
  5. Spread all of this on to the plates
  6. The colonies that grow are tetracycline sensitive cells (meaning their riboswitch is "off")