Field of Science

Responses to all the latest suggestions and comments

Preface:  To test the conclusions of Wolfe-Simon et al., I need to grow GFAJ-1 cells in phosphate-limited AML-60 medium containing 40 mM sodium arsenate.  Because the final cell density will be very low (limited by the low phosphate), to get enough pure DNA for the analysis I will need a large volume of culture, probably at least 500 ml.
At present the cells grow fine in medium without arsenate, but only occasionally grow in medium with arsenate, and so far only in small volumes.  When they do grow in arsenate they grow just as well as in the control cultures without arsenate.  So I suspect that there is some uncontrolled variable in my growth conditions that usually prevents growth in arsenate, but I don't know what it is.

17 Comments on the previous post:
Anonymous said...
Hello Rosie,
Do you have frozen or lyophilized stocks made from when you first received the culture? Can you start a new working culture from frozen stocks in case being cultured in the absence of arsenic causes the cells to become sensitive to arsenic?
Josh Neufeld (Waterloo)
Anonymous said...
If the cells are not pure culture, some may have plasmids conferring arsenic resistance plasmids.
October 18, 2011 12:08 PM 
Acleron said...
From someone totally uneducated in microbiology, but could the stock be impure and have a very small number of cells which can grow in arsenate?
michiel's suggestion would disclose this.
October 18, 2011 2:45 AM 

Yes, all the ±arsenate cultures I've done were started with tubes of the same frozen stock of GFAJ-1.  I grew and froze these cells from a single colony soon after I discovered that adding glutamate to the medium gave good growth.  These cells were grown in phosphate-limited medium for several generations before they were frozen, to deplete intracellular stocks of phosphorus.

 michiel said...
Dear Rosie,
Have you checked whether the bacteria in arsenate cultures are still viable? Wash away the arsenate move them to the a rich medium and see what happens.If the revive growth might just have stunted.
October 17, 2011 11:37 PM 
I've done this sometimes - plating the cells from arsenic cultures that failed to grow onto agar-solidified medium with no arsenate.  Usually there are still some viable cells, but knowing this doesn't help me understand why the cells usually don't grow.  

 Petri Dishing said...
As unhappy as the cells are in arsenate, could the arsenic itself been screwing with their motility in some fashion? That's the only explanation that comes to mind re: mixed v. still cultures.
October 18, 2011 1:46 AM 
Yes, it's conceivable that the cells in glass tubes failed to grow in the latest experiment because (i) the arsenate inhibited their usual motility and (2) they weren't being mixed.  But non-motile cells usually grow to fairly high densities in stationary cultures - it just takes them a bit longer because access to nutrients is slowed by the need to rely on diffusion rather than active mixing.

 Alexa said...
Maybe I'm violating a sacred boundary with this suggestion, but I would love to hear Felisa Wolfe-Simon's thoughts on this. She must have done something differently (her error bars in Fig. 1 are tiny!), and we're all tearing our hair out trying to figure out what it is. Have you considered just asking her? Is she too busy to try to help? As a scientist, I'm thrilled when I see that someone has reproduced my results, especially in a different lab. Wouldn't she also want to see this?
October 18, 2011 10:46 AM 
I'd be delighted to get advice from Dr. Wolfe-Simon.  But given my previous criticism I don't feel right about approaching her.  She certainly must know what I'm doing, so if she doesn't comment I assume it's because she chooses not to. 

 Anonymous said...
The cells have likely mutated, erasing any former capacity for growing in an arsenic-laced environment.
October 18, 2011 1:24 PM 
But sometimes cells inoculated from the same frozen stock have grown just fine.  One time I even took cells that had grown up in 40 mM arsenate and transfered them into medium that another inoculum had failed to grow in.  These arsenate-resistant cells did not grow in the new medium.  This suggests that the problem is with the medium, not the cells.  Of course it's always possible that I made a mistake in preparing the medium, but I've been very meticulous, especially lately as I became increasingly concerned about reproducibility. 

 GH said...
Is it possible for arsenate to interact with any of the components in the medium?
And have you been using the same ingredient containers all along? (i.e., have you finished off a container of something and started using a new container of that ingredient?)
The reason I ask: My experience with trace metal toxicity is that any time we opened a brand new ingredient container we had to test a whole panel of metal concentrations using the new ingredient(s). This is b/c the trace metals would complex with the various organic compounds in the medium ingredients. Since we had to use complex/undefined ingredients (such as casamino acids or yeast extract), the only way to guarantee that consistent bioavailability (and toxicity) of the trace metals would be to use the exact same ingredient containers. Same old container, very reproducible results. New container, new toxicity profile. 
So if you haven't done so already, try talking to a geochemist about whether there could be anything in your medium that could be interacting with the arsenate, either chemically or physically. This might explain why some batches of arsenate medium are more toxic than others.
If that line of inquiry doesn't pan out, then my next guess would be genetic instability of some type for the arsenate tolerance phenotype.
October 18, 2011 3:38 PM 
I've been using the same chemical stocks throughout.  I only have one bottle of 1 M sodium arsenate.  Some of the earlier experiments used medium made with an earlier batch of the '4x AML-60 salts', but all the recent ones have used the same batch.  I only have a single batch of the 500x vitamins mix and the 1000x trace elements mix - both are stored frozen and thawed and mixed when needed.
With respect to the genetic instability issue, see above comment about the failure of arsenate-grown cells to grow in medium prepared on a different day.

 Russell Neches said...
Do you have a chemostat? It might be interesting to get them going in continuous culture, and then slowly crank up the arsenic. If the problem is that they've adapted away from arsenic tolerance, that would be one way of trying to get them back on the wagon.
Even E. coli would start to tolerate arsenic if you took it slow enough. I suggest the following experiment :
[1] Sequence all available strains of GFAJ-1
[2] Grow them in chemostats under conditions that would drive them to arsenate tolerance
[3] When they are able to tolerate enough arsenate to satisfy you that they are adapted, resequence them.
[4] Align the resequenced genomes with the non-adapted strains
[5] Design primers to amplify up the affected regions
[6] PCR up environmental DNA from the original habitat with the primers, make some clone libraries and sequence them (also PCR up sequences from your lab strains, both adapted and non-adapted, just to make sure they work)
[7] Align the PCR'd sequences with the GFAJ-1 genomes you've got
If GFAJ-1 ever was arsenate-tolerant, you should see at least some similarities among the lab-adapted adapted strains and the environmental samples supposedly living in arsenate.
If not, I think we'll all have to notch up the skepticism another couple of pegs.
October 18, 2011 3:53 PM 
Interesting yes, but that would be far more work than this project deserves!  My goal isn't to create a new arsenic-resistant strain of GFAJ-1, but just to find the right conditions for the strain I have to grow reproducibly in arsenate.  It grows sometimes.... 

 Popi said...
@Alexa: Wolfe-Simon was "evicted" from her previous lab after all the chaos, so I guess she might very well have lots of free time to give advices..
Actually I think Rosie should hire her. This would give a really positive message about science and women in science.
October 18, 2011 5:00 PM 
She'd be welcome.  I don't have money to pay her, but I understand that she still has her NASA fellowship.

 NotAnAstrobiologist said...
This is a tough one...
What I would do in your place, start parallel cultures in -As and, say, 5mM arsenate...
Keep seeding cultures in progressively higher [+As], (and keep the old lower [+As] going as well)...
See if you can pinpoint the concentration where things blow up...
Presumably (based on your earlier results) -As should be able to keep growing normally without any problems...maybe at 15mM cells will start to die for magical reasons that can eventually be figured out.
October 18, 2011 8:43 PM 
I did an experiment with plastic and glass tubes a while back.  The cells in glass tubes grew fine in 10 mM arsenate and 40 mM arsenate, but the cells in plastic tubes didn't grow at all.  

 Paul Orwin said...
Our students at CSUSB presented this paper in Journal club, and we had, shall we say, a lively discussion :). One thing that struck me in reading the published back and forth in Science was the proposal that GFAJ-1 uses the high-affinity P transport system induced by arsenate. This was dismissed by FWS out of hand because in her mind it would lead to higher arsenate toxicity (I think that is what she was saying, I am not an expert on this). But it seems to me that if GFAJ-1 has a robust detox mechanism (reduction and sequestration or some such) it could induce the high affinity uptake and still handle the influx of arsenate. Further, it seems like the enrichment strategy they used would select for variants of exactly this type. This variant might be very unstable because expressing high levels of the detox mechanism would be a competitive disadvantage in the absence of As. The analogy that makes sense to me is something like a drug efflux pump. Several other commenters have already suggested methods to deal with this (going back to original stocks, using chemostat culture or serial increase in As) but the more i think about it the more this seems like the simplest explanation, and a testable one at that. Thanks for doing these experiments, this is really a nice way to show students and the public what science should and can be.
October 19, 2011 9:13 AM 
Sometimes the cells grow just fine in arsenate.  (Yes, I know I keep repeating myself...)

 Anonymous said...
Have you finally added tungsten to the media? Maybe tungsten is necessary for the high affinity P transport system induced by arsenate that Paul Orwin discussed above.
October 19, 2011 4:03 PM 
Yes, I added the tungsten to the trace elements stock well before I began trying to get the cells to grow in arsenate. 

 Anonymous said...
How about you throw the towel in and stop wasting your own time/money trying to make sense of FWS' bunk science. The paper should never have been published and in these times of tough funding you shouldn't be wasting your time trying to replicate her crappy work!
October 19, 2011 7:57 PM 
It's true that, from a strictly scientific viewpoint, there never was any justification for trying to replicate these results.  But strong 'sentiments' were expressed several places that someone should try anyway.  If I can't get reproducible growth I won't proceed - I wouldn't trust my results and I wouldn't expect anyone else to.

 Alexa said...
@Anonymous, 7:57 PM: The question is, at what point can everyone agree that Rosie has sufficiently demonstrated the "bunkness" of FWS' work? I didn't believe the growth curves even back in December, but what about the people who were willing to give them the benefit of the doubt? These people still exist--you can find them in the comments section of the PopSci article. And what about the authors of the paper, who apparently don't take seriously anything that isn't published in a peer-reviewed journal? I am also stumped as to how Rosie should proceed (at this point, would a journal publish her results?), but at least she is doing something to try to mitigate the rampant confusion and misunderstanding regarding this whole story!
October 20, 2011 7:53 AM 

 Anonymous said...
@alexa, I agree that Rosie should be commended for trying to make sense of the mess that is the FWS paper, but how far will this go? Seems like even the most basic of experiments (growing a culture) cannot be replicated reliably. And people are suggesting sequencing of all the different "strains" of GFAJ-1? Setting up a turbidostat? Funding is tough right now and the cost/burden of replicating/repeating this work ought to be on Oremland and FWS. It still blows my mind that Bruce Alberts hasn't done anything. If Oremland is a respected scientist he ought to try repeating (with better experimental design i.e. gel purifying DNA, MS/MS of the "As-DNA", etc) and retract the paper if they cannot replicate. Hats off to you for trying Rosie, but I hope your real research doesn't suffer from this distraction, although I must admit its been entertaining!
October 21, 2011 7:03 AM 

 Paul Orwin said...
I disagree strongly with the notion that Rosie should give up (although it's obviously her choice and not mine). Although I think FWS and colleagues misinterpreted their data, I don't think it was fraud, I think they found an interesting bug that they were able to enrich from Mono Lake and coax into growing under high As/low P conditions. The mechanism of that is interesting! I think that the As for P in DNA is incorrect, but that doesnt mean the whole thing is worthless. As an aside, I think any working scientist has to spend a lot of time thinking about practical concerns like getting your next paper published or grant funded, so side projects like this are obviously on a "shorter leash". And why would you think Bruce Alberts or Norm Oremland would want to do this? Finally, of course people in a blog comment section are going to suggest over-the-top solutions (it's alot easier to tell someone else to do something than do it yourself). But maybe someone will have a really great idea! You never know - if you have an idea, suggest it.
PS- growing a culture is not always "simple", and this set of experiments is good evidence for that.
October 21, 2011 8:30 AM  

No growth in 40 mM arsenate in ANY container!

And excellent growth without arsenate in every container...

As I had planned, I mixed the cells (about 10^5 cfu/ml) with 500 ml of medium, then split the medium in two parts, adding arsenate to one half and the same volume of water to the other.  Then I distributed the cultures among all the containers and put them in the 28 °C incubator for about 36 hr (sitting on the shelf, not agitated in any way).  (The biggest bottles, the orange-capped ones I mixed the initial cultures in, were in the 30 °C room.)

The containers with the blue '-' labels have no arsenate, those with the orange '+' labels have 40 mM arsenate.

The complete lack of growth of the arsenate cultures in the screw-capped glass tubes contradicts my previous results.  Might growth in arsenate depend on gentle mixing as well as on mysterious unidentified factors?  (This shouldn't matter since the cells are motile...)

This is ridiculous.  I don't think I've ever had such blatantly non-reproducible results before.  All I can think of to do next is to once again test growing cultures in glass screw-capped tubes, in medium with and without arsenate.  But even if the cells grow with arsenate in this next test, I won't have any idea why, or why they didn't in this experiment.  And anyway this experiment clearly says the problem isn't the containers.  

Readers, any ideas?

Yes, that last experiment was grasping at straws...

That last experiment was motivated more by wishful thinking that by having actually discovered why my GFAJ-1 cells grow inconsistently in arsenate.  Result:  cells in foil-covered glass flasks grew fine without arsenate, not at all with it.

Now I need to put on my serious-science hat and do a test of multiple factors.

Plan:  Prepare large volumes of two parallel mixtures of GFAJ-1 in AML60 medium, one with 40 mM arsenate and one without.  Then place replicates in many different culture conditions.  Plastic screw-cap tubes.  Glass screw-cap tubes.  Loosely capped glass tubes.  Loosely capped plastic tubes. Tiny tubes.  Big tubes.  Foil-covered plastic flasks.  Foil-covered glass flasks.  Screw-capped glass bottles.  Screw-capped plastic bottles.  Large volumes of liquid with little air space.  Small volumes of liquid with lots of air space.  Stationary. Gently rocking.  Shaken, not stirred.  Vigorously agitated.

Back in the lab, at least briefly

The last few weeks I've been in teaching-zombie mode, with my brains sucked dry by 350 second-year genetics students.  But I've only 4 more classes to go (each taught twice) and this morning I went into the lab and cleaned up all the glassware from the last three or four experiments.  Now I'm ready to start a new one.

This is a very unambitious experiment, the next tiny step in solving the "Why won't GFAJ-1 grow consistently in 40 mM arsenate?" problem.  In previous experiments the cells grew well with arsenate in small glass screw-cap tubes (now soaking in preparation for future re-use), but my DNA preps will need much larger volumes (500 ml or more), because cells can't grow to high density when phosphate is limiting (duh).  So now I'm going to try growing them in glass flasks with and without 40 mM arsenate.  I'll grow them on the communal shaker in the 30 °C room rather than in our spare shaker since our shaker is needed for other experiments.

So:  250 ml glass flasks (I have 6 sterile and ready to use), with 20 ml of AML60 medium with glucose, glutamate, and added phosphate at 0, 3 µM and 1500 µM, with and without 40 mM sodium arsenate.  All inoculated with phosphate-limited GFAJ-1 cells from my freezer stock, at about 10^4 cfu/ml.