Contact Dependent Inhibition (CDI) group page

Plan

1a. Growth

  • CDI-
  • CDI+ (2x)
  • resistant mutants (3x)
  • mixes: CDI- + res (3x), CDI+ + res (2x + 1x = 3x)

Everything in 2x plus blanks.
Team: the boys.
Status: CDI-, CDI+ (2x), resistant (2x) have run overnight, results below. CDI- replicate + mixes to be done.
Results: overnight growth of pure strains:
growth_curves_day1.pngdoubling_times_day1.png

1b. Resistance rates

  • inhibitors (2x)
  • parallel cultures (5x)
  • fluorescent plate reader :-)

Fluctuation test on resistant strains, but making sure they are there for a reason (not just "happened to never meet a CDI+").
Everything in 2x would be good (we can then pool statistics).
Team: the boys.
Status: Done.
Results: mutation rate for 49m is u ~ 10^-8, for 4945 u~10^-7.

2. CDI+ vs CDI-

  • titrate ratio CDI+ / CDI-: 1:1, 1:100, 1:10000
  • titrate cell density: 10^9 / ml, 10^7 / ml, 10^5 / ml (the latter might require some tuning)
  • when titrating cell density: take samples and measure cluster sizes under microscope
  • (later: develop model for cell density dependence)
  • (later: plus discuss toxin-independent cluster formation as an evolutionary strategy - might give a different angle on "selfish element" issue)
Everything in 3x would be good, or else 2x also ok.
Team: the girls.
Protocol :
Results :




3. Single copy plasmid

  • growth: 3 media (glu, gly, succ), 2 plasmid strains, CDI+(101), CDI-(105)
Team: JJ, M
Status: 7/30: prepared in above media, and growing.
  • cell density: see above but with 1-copy plasmid
Team: A, S.
Status: ongoing.
results:

4. The other cool things

  • time-lapse microscopy (plasmid + patterning):
Status: tested under microscope, results being analyzed.
  • sector formation: CDI+ / CDI-, mix of two CDI+
Team: the boys.
Status: unknown (please fill in).
  • competition assays with mixes of two CDI+
Team: M + JJ + S.
Status: started cultures overnight.
  • single-cell competition in microbubbles
Team: F, JJ, Nick.
Status: We got the bubbles and a single strain in it (RFP targets), and also both kinds (CDI+ green, CDI- red). The bacteria are taken from overnight culture, i.e. stationary phase:
2-9-14_together_test2.png
We also got them together in a movie over an hour, but there is bleaching in the GFP channel:


































The CDI+ bacteria are able to bind many CDI- and form tree-like structures:
competition_0.2dil_YFP_BFP-color-bg.png

We tried to grow the bacteria in the bubbles to observe non-stationary dynamics. It turned out to be more challenging than expected. We often get a lot of background fluorescence, so we used minimal medium + glucose instead of LB, There is still a large zoo of background noise levels, but the bacteria definitely grow a couple generations in the bubbles at 37 C:

BEFORE GROWTH
AFTER GROWTH
competition_0.2dil_YFP_BFP-24_c1+2+3-1.png
yGW992-10_c1+2+3.pngyGW992-11_c1+2+3.png
Results: droplets seem like an ideal environment to study single-cell competition dynamics of CDI without the complexity of 2D spatial structures of plates. There are, however, a few tricky points:
  1. background noise might be hard to control (if the bacteria leak out GFP or other fluorescent metabolites, either dead or alive)
  2. bleaching is an issue at single-cell level
  3. bacteria swim in and out of focus
If we were to refine the experiments, we would:
  1. get the right microscope filters (YFP, BFP)
  2. use chromosomal fluorescent inserts, with strong primers
  3. sort droplets at the end based on total fluorescence
The biological conclusions are:
  1. CDI+ bacteria are much more sticky and tend to have larger cells than CDI-
  2. one CDI+ bacterium can bind many targets, even during stationary phase
  3. the efficiency of CDI-mediated killing at single-cell level might be low