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Hypothetical experiment

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Hypothetical experiment

Postby Cat » Tue Dec 06, 2011 8:54 pm

I have a vague idea for an experiment. I would like your help in developing it into a workable protocol.

Here are the basics:

Approximate average human generation time is 20 years. For E.coli it’s 20 min at 37°C. So,

20 min ≈ 20 years
X ≈ 1,000,000 years
X = 1,000,000 min = 16,666.67 hrs = 694.44 days = 1.90 years

1. Take some of the E.coli (whole genome is sequenced) and put them into dialysis cup/jar in normal LB broth.

2. Set up three sets of dialysis chambers: a) control – regular LB broth; b) experimental chamber 1: LB broth + small amount of some chemical detrimental to E.coli in high concentrations (like salt); c) experimental chamber2: LB broth + small amount of some chemical neutral to E.coli and normally unused by it (like some sugar that it cannot process – if strain cannot utilize sucrose, than use sucrose). Make sure that all chemicals added can pass through dialysis membrane.

3. Change solutions in the dialysis chambers daily constantly increasing concentration of the additional chemicals in the experimental chambers. Calculate that at the end of two years concentration of the detrimental chemical in b) is double the lethal dose for this E.coli strain at the beginning of the experiment. For the chemical in c), concentration at the end of the experiment should be such that it would be the most abundant food source available in the solution.

4. Run the experiment for 2 years.

5. Inoculate from dialysis cups into fresh media (same as in dialysis chambers), O/N. Plate on agar/same media for isolated colonies, etc. Prepare DNA for sequencing. Sequence whole genomes of samples from all 3 experiments.

6. Test all isolates for ability to utilize chemical introduced in c) by growing all isolates (from all experiments) in minimal media with this chemical as the only food source.

7. Test all isolates for ability to grow in presence of chemical from b).

The way I see it, b) will be under negative selection while c) is under positive selection. My prediction is that b) will have less diversity than either a) or c).
Outcome of experiment c) will prove once and for all the impact (or lack thereof) of the positive selection on the population.

The major problem of this experiment is in logistics. How large the dialysis “cup” would have to be so that bacteria would remain in solution for the duration of the experiment? What else do I need to take into the account?

I would appreciate your input.

Thanks.
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Postby canalon » Wed Dec 07, 2011 3:27 am

I would suggest studying the material and methods of the experiment run by Richard Lenski...
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Postby Cat » Wed Dec 07, 2011 8:25 pm

That experiment describes conditions reminiscent of what I propose here to be for a “control” sample. Meaning there is no pressure and no incentive for development of new traits. This leads to differentiation just as they observed.

I am more interested in trying to simulate natural mode of evolution where environment is not static. Also, they remove 99% of the culture each day thus limiting possibilities and eliminating most dead bacteria. I want to maintain all bacteria including those dead because theoretically this would increase possibility of lateral gene transfer.

If you noticed they did not observe any significant gains in the genetic material (http://www.nature.com/nature/journal/v4 ... 480-s1.pdf). And that would be consistent with the hypothesis that gains such as duplications are either caused by interventions (like viruses), when organism is on the edge of extinction (like whole genome duplications), or by lateral gene transfer.

Let me know if you have any other ideas.

Thanks.
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Postby canalon » Thu Dec 08, 2011 1:54 am

You will not be able to keep all bacteria dead or alive for 2 years. You have to remove some constantly. The best you can do is to maintain your culture at the edge of saturation (just run the calculate the number of bacteria at the end of the first day ;) ). That can be done in a chemostat, and there are plenty of size available. But they are time consuming to set up, can be expensive to run but when they work, they are great.
They can also be used if you simply want to keep your solution stationary in fresh medium, with just a filter to prevent the old medium to take the bacteria away. The conditions might be tricky to set, and you probably do not want anything as rich as LB if you want to avoid problems.
Another thing is that if you have only one strain of bacteria, just like in Lenski's experiment, the probability of HGT will be low, and even if it occurs, might not be easy to detect. Moreover if you use E. coli that is not naturally transformable in LB (but might be in other media).
How will you simulate phage? External DNA input (As a fecal bacteria, the normal place of living of E. coli is not all by itself, and neither in a rapidly dividing state, and very likely in a biofilm rather than free floating.

My 2 cents.
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Re:

Postby Cat » Fri Dec 09, 2011 1:41 am

canalon wrote:
you probably do not want anything as rich as LB if you want to avoid problems.



What sort of problems?
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Postby canalon » Fri Dec 09, 2011 2:21 am

Basically LB is not representative of natural conditions for E. coli. If you keep feeding a very rich broth to a population that is limited in size (but not in nutriment, you might have some weird things happening in you chemostat, and I wonder if the culture is going to be very stable.
Plus probably there will be some massive biofilm formation and contact inhibition, and the behavioour will be quite unpredictable. Anyway if you do not remove the cells as you go, you will never have the predicted number of generations, As in a saturated environment, the bacteria will stop dividing.
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Postby Cat » Fri Dec 09, 2011 3:53 pm

Actually I was thinking about that. What would happen if bacteria run out of space but not nutrients? I have no idea. Under normal conditions it would run out of nutrients or accumulate toxic waste long before it runs out of space...

As to LB, I am not trying to mimic conditions natural to E. coli. I am trying to create conditions mimicking nature - changing environment where negative or positive pressure is present and mounting. I am afraid that using minimal media predisposes bacteria to gene loss in the long run (you can see it in Lenski experiment). So maybe LB is not the best media and E. coli is not the best organism for such experiment. Maybe soil bacteria would be better. Can you recommend any (whole genome sequenced) such bacteria?
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