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Simplify, Simplify…

DNA seems to have two main threats to its well-being once it’s extracted and purified.

  • Nucleases
  • Spontaneous Hydrolysis by water

Nucleases are the big one that everyone seems to mention. The seem to be fairly sturdy enzymes, and they’re everywhere (including fingertips – hence the need to wear gloves whenever you get near DNA samples…), and they “eat” DNA rapidly. Theoretically, you can destroy the enzymes with enough heat, but you still need to worry about them getting in every time you pop open your sample to get some out.

Apparently, DNA even in pure water can tend to slowly fall apart spontaneously. It doesn’t happen very fast, but bit by bit, it can undo the links between the individual nucleotides.

A common way to try to deal with nucleases is to add EDTA to the solution. Nucleases need magnesium ions dissolved in the water to do their job, and EDTA tightly binds to magnesium (and calcium). The idea is to “use up” any stray magnesium ions in the solution so that even if nucleases get in, they’re inactive because they have no magnesium available. That’s why you see EDTA in the recipes for so many DNA-related solutions. Of course – EDTA doesn’t permanently bind up all the magnesium – there’s always a tiny fraction that stays in the solution. So, although EDTA can drastically slow down any nucleases, it won’t actually stop them.

There are also some interesting chemicals which can be added to destroy all proteins (including nuclease enzymes). Guanidine Thiocyanate is one rather nasty chemical that does this. 2-mercaptoethanol is another. Various other detergents like CTAB may also denature any proteins. Since they don’t harm the DNA in the process, you could keep the DNA sample dissolved in a solution with these chemicals…but then you can’t do PCR with the sample as it is, since the protein-denaturing chemicals will also destroy any enzymes that you WANT, like DNA Polymerase, when you try to mix it into your reaction.

I think the latter option will be great for collecting field samples (in fact, it’s papers specifically on the subject of preserving samples in the field with CTAB and Guanidine Thiocyanate based solutions that I’m adapting from), but isn’t going to be real useful once I’ve got my DNA relatively purified. What to do, what to do…

Actually, I think the answer’s simpler than I originally expected. I’ll just dry the purified DNA out. No water – no hydrolysis…and no nuclease activity, either.

I could actually just leave it as a dried pellet in the bottom of a microcentrifuge tube, but that leaves the problem of taking only a little bit of it for processing rather than taking the whole thing, and I want to avoid reconstituting it and re-drying it repeatedly. I think a variation of the “dry the DNA on a piece of paper” process will be in order – then I can just cut off a small strip of the paper to get a portion of the DNA. It appears that you can actually dunk the DNA-impregnated bit of paper right into whatever solution you’re using (like a buffered polymerase-and-primers solution for PCR) and go for it.

Among the several references I found on this, here are two:
Kawai J, Hayashizaki Y: “DNA Book”; Genome Res. 2003 13: 1488-1495
Burgoyne LA: U.S. Patent #5496562 “Solid medium and method for DNA storage” (1996); U.S. Patent and Trademark Office, Washington D.C.

Is GFP GRAS?

Woohoo!

One of my longstanding questions has been, is Green Fluorescent Protein safe to put in food?

I always figured that it SHOULD be – it comes from jellyfish, which I know people eat in some cultures (though I’m not sure if any of the ones eaten actually express GFP).

Well, it seems someone in 2003 did a proper test…Check it out!

Sure, this is still some way before the FDA declares it “Generally Regarded As Safe” but it’s one step closer.

Soon, my dream of genetically-engineered “Glogurt®” will become reality! AH, HA HA HA HA HA!

Okay then…

My summer classes are finally over. Got an “A” in immunology (go, me). Now I just need to make sure everything’s done next semester. I’ve already signed up for the last two Underwater-Basket-Weaving-type “General education” classes required at this college: Intro to Philosophy and “History of Western Art”. I also went ahead and signed up for Environmental Chemistry, too – it’s not required, but it’s one of the last “not required but useful if I have time for it” classes on my list.

Meanwhile – is it just me, or is DNA some obnoxiously fragile stuff when you don’t want it to be? Sure, leave a few flakes of skin or hair follicles at a crime scene and they’ll nail you weeks or months later, but try to “gel purify” some DNA and it just falls apart…

The samples from my last post, about the colony PCR of my Lactic Acid beer-bacteria, I cut the bright bands of presumably-16s rDNA out of the gel and ran them through one of those canned “gel purification kit” processes. Then I froze them until I had a chance to finish my classes and play with them.

Yes, I was wearing gloves. No, I didn’t lick the gel. I think I must have looked at them too closely or something and they just disintegrated out of spite. In any case, my attempt at a restriction enzyme digest turned up NOTHING (other than the “ladder” lanes) on the gel.

I’m beginning to really distrust canned kits. On the upside, that means I get to learn some more in the process of developing my own replacement protocols.

I will probably try re-amplifying DNA from the frozen samples and see if there’s anything at all left in there that can be saved. Otherwise, I’ll also check and see if the plates I made a few days ago still grew okay.

In other news – I’m toying with the idea of literally begging for my own microscope and home-microbiology lab equipment. As in, actually putting on a lab coat, taking an old hat, and sitting outside of scientific meetings and such with a cardboard sign saying “want my own microscope – please help”. Of course, I’d have to report any donations as “income” for tax purposes – I doubt they’d let me form a 501(c)(3) corporation dedicated to just buying me toystools for my own microbiological amusement.

I haven’t decided, but it’s under active consideration. It’d make for some interesting blogging (and I promise in return that I’d account on the blog for any money donated, and blog all uses of the equipment under Creative Commons terms so everyone can use it). It’d presumably take a while for this to get anywhere if it ever did – it seems it’ll cost about $400-$500 just for a (good) basic light microscope, plus another few hundred for a darkfield condenser and related upgrades. Plus, of course, me wanting to build some LED-based lighting for fluorescence microscopy ($500 canned commercial upgrade? Bah!). Incidentally, it seems Green Fluorescent Protein fluoresces best right around the wavelength of a typical, inexpensive, off-the-shelf ultraviolet LED…

And then of course I need a pressure cooker and one or more incubator setups and some petri dishes and trips to the grocery store for growth media and staining supplies and slides and… well, anyway, as much stuff as I can arrange to get. But the microscope is the one component that is unavoidably expensive.

Oh, yeah, and some space to keep cheese and beer culture organisms and such for later use…

Comments, anyone? Suggestions?

I, for one, WELCOME our new radiation-eating fungal overlords…

Though I am getting a little annoyed at the breathless prose about how it’s “like photosynthesis” and might be a way to sustain astronauts during long space flights and so on.

The story’s about a fungus they found growing (thriving, even) inside the reactor at Chernobyl, despite all the radiation the fungus is exposed to in there.

What the original paper – which you can find here from PubMed Central (and where you can find what the study actually shows, rather than the somewhat lower-content hype found in most news reports on it) – seems to show based on my hasty undergraduate-level reading is that the fungi do grow faster when exposed to “ionizing radiation”, and that it appears to be due to melanin in the plant getting energy from the radiation (and passing that energy on to the fungus to use for growth).

This is actually pretty spiffy, but really – so far – doesn’t look like “photosynthesis” at all. They’re not testing for any kind of carbon fixation, and I’m guessing that if there is any carbon fixation going on, that it doesn’t generate oxygen in the process. It also seems unlikely to me that even then, the fungus can grow autotrophically. This would seem to drastically reduce the possibility of this stuff ever being Purina® Astronaut Chow – you’d still need some other way to get the carbon dioxide out of the Astronaut’s air and put oxygen back in it. If you’re going to do that, you might as well just use plants (or cyanobacteria) and eat THEM.

Still, the implication that you could adapt some melanin-producing fungus to absorb “radiation” and turn it into useful materials of some kind is spiffy, even if it’s not going to allow us to turn nuclear fission plants and spent nuclear fuel depots into fungus-powered anti-global-warming-gas powerhouses.

One thing’s bugging me, though. I obviously don’t have enough understanding of how “ionizing radiation” behaves at a biochemical level, since I’m wondering if it’s proper for everyone to treat “radiation” (both from flying electrons and from high-energy light) as some sort of generic substance, whose only useful attribute is how much energy it has.

As far as I know, most of the “radiation” that the fungus inside the Chernobyl reactor is getting is Gamma-radiation – basically high-energy light (one step above “X-rays”, two steps above sunburn-causing Ultraviolet light). What the researchers are hitting their test-subjects with looks like it’s mainly “Beta”-radiation (which is to say – electrons)*. In both cases it’s “ionizing” radiation, which is to say (more or less) that the radiation knocks electrons off of atoms that it runs into in both cases, and in the ideal “spherical horse” world of a Physicist, the same amount of energy is going to knock the same amount of electrons loose from various molecules and therefore have the same effect, right?

Except I’m having trouble convincing myself that’s a valid assumption here. The results seem to show that exposure to radiation is somehow resulting in the melanin in the fungus being able to “reduce” a chemical (changing “NAD+” into “NADH”) that can potentially in turn dump electrons into the beginning of the Electron Transport Chain to in turn provide biological energy in the form of ATP…

Can one reasonably assume that the mechanism by which this happens would be the same regardless of the form of ionizing radiation? The big deal with melanin seems to be that it absorbs a wide range of light wavelengths (which is why it looks black to dark-brown, and why it protects skin from Ultraviolet radiation…) which implies that absorbing the gamma radiation is where the energy is coming from that makes the fungus thrive in the Chernobyl reactor building. I guess I’m just having trouble picturing how a much more massive, slower-moving electron could have precisely the same effect as a virtually massless, much faster photon. (Yes, I know that beta and gamma radiation are said to have the same amount of “effect” on living tissue per unit of energy…)

Is it possible that the melanin is directly “capturing” the beta particles (electrons), while gamma radiation is kicking electrons off of something ELSE, and melanin is then only indirectly taking up those? For that matter, is it possible that in both cases it’s just something silly like the radiation inducing hydrolysis of water, and it’s just hydrogen gas supplying the reducing power? Thinking about this is making me feel dumb – can anyone reading this explain what I’m missing here?…

I suppose I could just cheat and ask someone in the biology department. We’ve GOT a professor who ought to know – her research has specifically focussed on zapping prokaryotes with “ionizing radiation” (electrons from the college’s linear accelerator)…But that would rob my dear readers of the chance to participate here…

* – okay, it’s probably even more complicated than that. If I understand what the paper is describing and what my Minister Of Funky Physics Knowledge showed me, the source of the “ionizing radiation” for the experiments is radioactive Tungsten(W) and Rhenium (Re) (A “188Re/188W Isotope Generator”). W-188 gives off beta particles when it decays to Re-188. But Re-188 can go through some sort of funky subatomic rearrangement before it decays so that it can EITHER give off beta particles OR gamma-rays as it decays down to stable Osmium-188. I have no idea what the proportion between beta and gamma is at that step (the “conversion efficiency”) so it’s possible there’s enough gamma radiation coming out to do something, regardless of what the beta particles are doing. (The experiment doesn’t do any comparisons with “pure” gamma radiation, which I imagine is not simple to arrange…). So now I’m even MORE confused. Thanks, physics. Thanks a lot.

E.coli – the “Microsoft” of the biotech world?

…by which I mean, it’s not always the best tool for the job, but everyone insists on always using it anyway, and has a variety of excuses for doing so…

Honestly – I’m trying to set up a clone library of 16s rDNA sequences using this kit. Never mind which kit it is – it actually does seem to work. I was just struck by the amount of hassle involved in shipping and storing the kit and it’s supply of “competent cells”.

When you get them, take them out of the dry-ice they’re shipped in and put them in the -80°C freezer immediately or they’ll die! Only thaw them carefully just before you use them, and do it on ice or they’ll die! Don’t heat-shock them for more than exactly 30 seconds or they’ll die! Once you’ve got them growing, you have to keep moving them to fresh selective media frequently or they’ll die! Or, you can carefully place them in the -80°C freezer…or they’ll die! Don’t look directly at them or they’ll die! (Do Not Taunt HappyFunCell!…)…

Seriously, running those gigantic -80° freezers can’t be cheap. Wouldn’t it be more convenient if you could grow up your transformant as an ordinary culture and just add your DNA samples and some kind of inducer chemical to make them take it up? Surely there must be some other organism that might be made to work like that.

Actually, it seems a number of the “Gram-positive” (firmicutes) organisms can enter a state of “natural competence”, where they naturally take up double-stranded DNA molecules from the environment. Bacillus subtilis is one. I’ve even seen references to “natural-competence” based protocols for transforming B.subtilis (or other Bacillus species, presumably) but it only seems to be in an out-of-print, $400 book.

Wouldn’t that be more convenient (using B.subtilis that is, not the $400 book)? Plus, when you wanted to store your transformed culture for later use, you could just heat the culture up to, what, about 55°C for 15 minutes or so (as I recall) then let it dry. The spores will contain whatever “bonus” plasmid DNA you added (if spores didn’t keep plasmids, then anthrax wouldn’t be such a danger…) and will last practically forever at room temperature. Mix the spores with some dried nutrient powder and seal them in a foil packet. Instant transformants, just add water!

But NOOOOO…..”But, everybody else uses E.coli, so I have to.” “They only make ‘BogoGen SuperMiniUltraKlone Kit 2000’ with E.coli, and we have to use that!” “But, nobody knows that other stuff, but everybody’s already familiar with E.coli!” “I’m a BogoGen Certified E.Coli Engineer, and I say everything else is just a toy and doesn’t work!” “All the books and stuff are about E.coli…”

Bah! Pathetic excuses. Anybody got a huge wad of venture capital to throw at me? The more I think about this, the more I think ‘untapped niche’…Heck, the electricity savings on not having to run a -80°C freezer constantly alone ought to qualify for a good “Fight Global Warming – Say ‘No!’ to E.coli!” marketing campaign…

Bonus perk: All the natto you can eat…

Search Queries That Came To This Site: Part 1 – comic relief

But first – a quick notice: I just added a “rating” bar for posts. Feel free to vote – the more feedback I get, the more likely it is that I may eventually learn to write more consistently coherent and interesting things…

At this point, this little blog seems to get most of its meager traffic (by far) from search queries. The searches have been piling up, and I figure it’s about time to do some posts to try to address those searches.

For part 1 here, I believe I’ll start with the oddball searches which often don’t seem to have anything to do with microbiology or, indeed, sometimes anything coherent at all. It’s late, and I could use some comic relief. (In Part 2 I’ll discuss some of the unexpected-but-coherent searches that led to my blog, and in Part 3 I’ll post about the kinds of microbiology searches I kind of expect to see in the logs that I’ve gotten…)

Why MSN loses to Google and Yahoo:

  • Out of the 5 whole MSN queries that have led to this site, two of them are: “mazda” and “debt”. I have no idea why. (In fairness, the other three queries were perfectly plausible microbiology-type queries).

Just plain “Huh????”

  • Someone in San Jose got here by Googling the phrase: “Type of fruit makes balloon grow bigger”
  • Someone from Nairobi(?) got here by querying “death and nuisances”
  • From a Washington State school organization of some sort: “a powder that looks slimy looking when lemon juice is added”
  • From a Toronto school network: “how does pink solution work(remove stain)”. (Actually, they may have been looking for information on Eradasol™, which is a seriously nasty-smelling detergent/solvent of some kind which does a good job of removing microbiology-type dyes from floors, countertops, fingers, etc…)
  • From the UK: “in search engine type cell a room” (Uh…what?)
  • From Indonesia: “expired of natto” (are they trying to find out when you throw away Natto instead of eating it, or people who died from eating Natto?)
  • From the Department of Education in Orange County (California, presumably): “water ballon splater”[sic]
  • From the Department of Education in Queensland, Australia: “why does this material work for the room”
  • And finally, my personal favorite from (apparently) Google itself: “iron chef cheese balloon”

BLASPHEMY!

  • Both New Zealand and the UK got here trying to find out about how “mushrooms are evil”. This is completely unfounded – Mushrooms are our FRIENDS.

Kinda Scary

  • From the Vancouver area: “world’s best bathrooms, microbiologically” (Ah, but best for what purpose?…)
  • I got two different queries (both from Pennsylvania?) for “eating expired jello” (Actually, as far as I know, so long as the stuff remains dried in its sealed pouches, it’s probably safe to eat almost indefinitely. I’d be a little leery of expired pre-made gelatin, though – that stuff’s a relatively simple protein mixed with lots of water and, often, sugar. Sounds like very attractive food for microbes of all kinds, including some that might make you sick…)
  • Speaking of which, someone at University of Michigan was looking for “eating expired bread spore”
  • Someone from Illinois was looking for “old interrogation room pictures”(?!) on Yahoo…
  • Someone on a military base in Ohio somehow got here looking for “solicitation can be released at least how many days”

And, perhaps scariest of all:

  • someone in Alabama had an odd search phrase: “organism +I*”

Why is this scary? Everyone remembers Isaac Asimov, who (while he was a live organism) wrote “I, Robot”, right? Well, obviously this means that a secret cabal of government agents managed to steal Asimov’s brain and upload it into a computer, thus creating a Robot Isaac Asimov (and this searcher wanted to know when Robo-Asimov would be publishing “I, Organism”.) Obviously, government “working” as well as it does, their Robo-Asimov still uses “Reverse Polish Notation”, hence the reverse-entry of “Organism I”…Okay, enough silliness for one evening. More – hopefully – tomorrow.

Sorry about the recent case of blogstipation…

So, here I am blogging from the hospital…

What? Oh, no, I’m fine, it’s just right across the street from where all of my classes are this semester, and they have a fairly decent cheap cafeteria. Plus, if I get this particular table, I can just barely get enough of a signal with my laptop’s external antenna to connect to my college network account.

Last week was spring break. Although I probably SHOULD have spent it drunk and naked, according to common wisdom, I instead spent it trying to catch up on sleep and doing a bit of culturally and educationally enlightening travel.

Aside from yesterday’s trip to the Opera, we managed to get out to visit Lehman Caves. As one might guess, I was hoping I’d get to find out something about the microbiology of the cave (in addition to ogling the impressive mineral stuff.)

As far as the microbiology goes, I was quite disappointed. One of the small books in the visitor’s center mentioned the existence of chemolithoautotrophic bacteria. In one paragraph. The entire content of which I just summarized here. Not even an identification of what kind of bacteria they are. The guide for the cave tour only knew that the bacteria in the cave were “harmless” (well, yeah, I kind of imagined they would be). There were also cyanobacteria happily if slowly growing near the lights, which nobody seemed to know too much about either.

I did get the name of the person responsible for issuing research permits – I’m seriously considering trying to make the cave one of the sites for my Senior Thesis study.

I did some other things, too, but I’ve got to pack up and head for class now. In case anyone besides my immediate family is reading this regularly (please comment if you are!) I will try to post a lot more often now – the last couple of weeks have just been a major distraction.

This is your brain. This is your brain on Microbiology…

In an effort to eat a relatively healthy diet, I occasionally eat pieces of wholesome, natural fruit.

There, I’ve admitted it. I can no longer live the lie that I only eat junk-food. Of course, to maintain some appearance of having a normal mainstream type of diet, I at least tend to go for the pre-cut fruit mixtures – I’ve got way too much going on to have time to prepare cut fruit salad from scratch.

Anyway, a few weeks ago I had a platter of these, still sealed in their plastic container, and didn’t get around to eating them in time. About a week after it had expired, when I went to throw it away, I saw a few little white lumps growing all over the pieces of fruit. Non-fuzzy, so I expected they were bacterial rather than fungal. Of course, there’s only one thing I could think when I saw that.

“Oh, wow! It looks like there were only about 5-8 bacterial CELLS on each piece of fruit when I got them! Those things were really CLEAN!”

(Of course, once you can SEE the colonies growing, there are a lot more than a few cells there. Each visible colony’s probably got millions of the little buggers, but each colony starts as a single cell.)

This was, of course, followed by me lamenting that I didn’t have some culture supplies and a microscope of my own to examine them with. Sigh. Anybody out there have any extra microbiology equipment you’d like to donate to a good cause?

So, what’s next? Should I try to start a series of bacterial taxonomy posts? Searches for “what’s a gram-positive?” and “what’s a gram-negative?” sorts of questions seem to be popular ways to reach this blog…

I am filled with shame…

It’s been a long day away from home, and I’ve got nothing much prepared for tonight – the last day of “Just Science” week. (Not that I’m going to stop posting after today or anything…)

So, I’ll cop out, and instead post a question.

What textbook(s) are you currently using for Microbiology classes, and what do you think of them? My “Introductory Microbiology” class was over 8 years ago, but the textbook was Tortora, Funke, and Case – “Microbiology: An Introduction (sixth edition)”.

I found it annoyingly heavy on the “disease-listing” and way too sparse on the rest of the microbial world – though they did have a couple of chapters on applied/industrial type microbiology.

Please leave comments…

My posts will likely be pretty sparse until after Wednesday, when I have back-to-back Microbial Genetics and Pathogenic Microbiology exams. Ick. As you can imagine, I’ll be studying a lot for the next few days to make sure I’ve learned what I’m supposed to up to this point.

Cheap Miscellany

Tonight’s post will be a bit short, but I’ll try to make up for it tomorrow. (Three more days of Just Science week to go…)

First, a quick unsolicited plug for someone else’s site: Aetiology is doing a series of posts on “Normal Flora” – that is, the microbes that normally live on and in healthy people (as opposed to microbes that just cause disease). Since that’s an area where my own interests overlap with the more conventional “medical” microbiology, it seems appropriate to mention it here. As of right now, there is a Part 1 and a Part 2. Interesting and informative stuff.

Second, a small addendum to the previous post – I mention that I think when people say “Gram-Positive” they generally really mean Firmicutes, but I just realized there’s one exception to that. The Firmicutes actually include Mycoplasma and related bacteria – which have no cell wall at all. I would guess their ancestors were normal “Gram-Positive”-type firmicutes but somewhere along the way “lost” function of a key gene involved in making the thick cell wall. (There’s a similar group of Archaea – the Thermoplasmata. ) These don’t stain Gram-positive (and perhaps don’t stain gram-negative, either – seems like such fragile things would be destroyed by the staining procedure). In order to see these in the microscope, the standard method seems to be to use a chemical that actually stains DNA instead. Instead of staining the outer surface of the cell like most of the classical stains, this stains the inside of the bacteria (which tend to have their DNA spread more or less throughout the entire cell in one form or another, since they have no nucleus to pack it into) with a fluorescent material, which you can then see in the microscope with the right kind of light. You can also use this kind of technique for other bacteria, too. The “Live/Dead” stain I previously mentioned works this way, if I remember correctly.

Since you probably don’t want to try heat-fixing Mycoplasma, you have to use a “fixative” (a preservative made of alcohol and pure acetic acid) and then air-dry the slides.

This leads to one last correction – in a previous post I suggested that it was unlikely that you could “glue” your smear to the slide rather than heat-fix (assuming that the “glue” would interfere with the subsequent staining and viewing of the sample) – this is actually not completely correct. I heard today of a protocol for doing endospore and acid-fast stains which called for mixing the culture sample with serum on the slide to make it stick, so there are at least a few ways to “stick” cells to the slide and still look at them.