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.

Argh! Blogstipated again!

I actually am still here, but Spring 2007 finals went straight into Summer 2007 classes, and it’s pretty intense. A six-week Immunology and Immunology lab alongside an entire semester of “Speech” class crammed into 4 weeks means I’m pretty frantic at the moment.

Although the semester is over and the paper turned in, I am still working on my 8 beer-bacteria isolates. In fact, re-doing “colony” PCR with a few µl of broth culture seems to have actually worked pretty well:

To the left, there, you can see the results. Interestingly, if you look just to the right of where the “wells” are in the gel, in several lanes you can make out a visible band of obviously large DNA molecules, which I presume are genomic DNA from the bacteria. The fact that they seem brighter in the lanes where the 16s band at the end (or at least, I’m ASSUMING that’s what that is, and my reaction worked) is dimmer tends to support my suspicion that I’m “swamping” my reaction with the template DNA and making it hard for the reaction to work sometimes.

Anyway, once I’ve worked out how to actually do a useful Restriction Enzyme digest to get an idea of how many of my isolates are really unique, I should hopefully be about ready to get them sequenced so that I can identify them. Then, I’ll be ready to try them out on my thesis project. Meanwhile, both my current DNA extraction from my recent thesis project sample and the amplified 16s gene bands for my lactic-acid bacteria from the beer are sitting in the freezer.

I’ve got an intense couple of days now, but hopefully I’ll have time to blog a bit more over the weekend.

Colony PCR – because DNA extraction protocols suck.

If you’ve got a culture of a single type of bacteria and you want to identify it, the standard method is to figure out the sequence of one particular gene, the 16s rDNA gene. That is – it’s the gene which encodes the a piece of RNA that gets used by the ribosome in part of the process of “reading” which amino acids to link together to make a particular protein. This is something that every prokaryote known has, and parts of it are conserved, so they’re similar enough to compare, while other parts can vary a lot, providing enough “difference” to tell different organisms apart.

To figure out the sequence, you use PCR to “amplify” this particular gene, making lots of copies of it so that the sequencing machine can clearly see the signal from each part of the sequence. And before you can do that, you have to get the DNA out of the cell relatively intact.

That part can be a pain. There are lots of different ways people have come up with (and made special canned “kits” out of) – you can use chemicals to try to dissolve the cells and let all their guts (including the DNA) out, you can try to mash them up with tiny glass beads in a “bead-beating” machine, you can stick them in a blender, you can even just boil them for a while…then usually you go through several steps of centrifuging and mixing with different chemicals and then centrifuging again until you’ve hopefully finally got the DNA out and gotten rid of most of the other cell bits. And, hopefully, you haven’t accidentally chopped up the DNA too much to use in the process.

Fortunately, there’s a trick you can sometimes use, referred to as “Colony PCR”. In it, you literally just touch the top of your colony of cells and shake them off directly into the PCR tube. Then you just include an extra 5-10 minutes of 95°C heating to hopefully cook open enough of the cells to release DNA (and cook the cell’s enzymes to death so they don’t degrade the DNA and interfere with the PCR).

Not real reliable if you’re trying to do anything quantitative, like trying to figure out how many copies of a gene are in each cell, or trying to get an accurate estimate of how many cells of one type or another are in a mixed culture, but if you just need as much of a particular bit of DNA as you can get – such as for sequencing – a lot of people use this.

I just tried it on my Lambic isolates. Two of the 8 bacterial cultures worked beautifully. I’m pretty sure the problem with the other 6 was just the sheer amount of bacteria I ended up adding to the reaction – too much seems to “swamp” the PCR process and keep it from working. I’ll try it again this week. But it does seem to indicate that it works, at least.

Tasty Acids

The story so far – I’ve got 8 live bacterial cultures (and two yeasts) obtained from a bottle of Peach Lambic, imported from Belgium. I strongly suspect that 6 of the 8 are Pediococcus species, and the other two are in the Lactobacillus genus. It is also possible that some of them might turn out to Leuconostoc or some other genus, but I suspect them all to be in the Order Lactobacillales somewhere, anyway.

Hopefully I’ll be able to get good, definitive sequence data from the bacterial isolates later this week.

Lactic acid seems to be the predominant acid in Lambic ales, produced by the various bacteria which break down the sugars in the beer and spew out lactic acid as a waste product.

Pediococcus also shows up in wines, where it’s associated with “malolactic fermentation” – where it converts the harsher malic acid into the more mellow-tasting lactic acid.

Thinking about this led me to think about the other distinctive acids found in foods. Here’s a listing (in no particular order) of some, with foods associated with their distinct flavors:

  • Lactic acid = “Yogurt” acid (and Sour Cream.  And many types of pickles.)
  • Malic acid = “Apple” acid (“Green Apple” flavor)
  • Tartaric acid = “Grape” acid (Verjuice and “Grape flavor”)
  • Acetic acid = “Vinegar” acid
  • Citric acid = “Lemon/Lime” acid (or “Pixy Stix®” flavor)
  • Propionic acid = Swiss cheese acid

In other news, I need a real microscope of my own.

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…

More Lambic pictures

Ah, that’s better – a more traditional heat-fix/simple stain (using Methylene blue) shows my yeast isolates better:

Sally the maybe-Brettanomyces-type yeast
(“Sally”, a yeast that I suspect is a Brettanomyces-type yeast.)

Sam the...Saccharomyces-type yeast?
(“Sam” now looks awfully small…but more experienced observers than I am said that it could actually be a Saccharomyces-type yeast.)

Lucy the possibly-PediococcusI also got two more Coccoid-Cluster-type Gram-positive bacterial isolates. The look pretty much the same under the microscope, though one had gooey wet, slightly larger colonies than the other’s smaller, hard-lump colonies. I see another one of those tetrads in the hard-lump-colony microscope image.

All told, I now have 10 isolates to check out. I’ve been given the go-ahead to try sequencing on the 8 bacterial isolates so hopefully I’ll be able to get a clear identity for Fred, Sid, Lisa, Lucy, BillyBob, JimBob, BettySue, and MarySue. Sally and Sam will have to wait for now, though I’m looking into ways to characterize them, too.

“Live and active cultures” – of beer.

I’ve got a project going to isolate as many yeasts and bacteria as I can from the dregs of a bottle of relatively-famous-brand Lambic ale.

So far, I’ve got at LEAST 3 different types of bacteria and two different yeasts – all of which I suspect are “intentional” – that is, the bacteria are probably lactic-acid bacteria (Lactobacillus, Pediococcus, etc.) which are expected to grow there, and the yeasts I believe to be a Brettanomyces-type yeast and a Saccharomyces yeast (based purely on what I expect to find and the small amount of microscopy that I’ve been able to do so far.)

I have at least one and maybe two different “Gram-positive” rod cultures which I believe to probably be Lactobacillus species. I have several isolates of generic “clusters of Gram-positive coccoids” of which there are at least two different types (which look more or less identical in the microscope, but one of which seems to generate acid while eating mannitol and one that doesn’t).

I have so far named three isolates from Sabouraud agar: Sally, Sid, and Sam.

Sally the Yeast
Sally, the maybe-Brettanomyces-type yeast – 400X magnification (Lactophenol Cotton Blue stain)

Sam the Yeast
Sam, the maybe-Saccharomyces-type yeast – 400X magnification (Lactophenol Cotton Blue stain.)

Sid the [lacto?]bacillus-type-thing
Sid, presumably a Lactobacillus-type bacteria – 1000X magnification (Gram stain)

I’ve also collected four isolates (which may actually just be two different organisms) from an initial inoculation on MSA – BillyBob, JimBob, BettySue, and MarySue. MarySue is the one that seems to be “fermenting” the mannitol.

BillyBob, maybe a Pediococcus?
This is BillyBob (I clipped part of the image and moved it closer to the little “ruler”). The others look essentially the same when Gram-stained.

I’ve also got a bacillus-type (presumably Lactobacillus) critter that showed up on an initial BHI which may or may not be the same as Sid, and I got two more BillyBob/MarySue type colonies on another MRS agar plate.

Interestingly, when I did the original inoculations, it’s the ones that I added the LEAST amount of beer sediment to (20?l) that seems to get the growth – higher amounts may just add so much sugary solution (this stuff is quite sweet) that it inhibits growth.I really hope I can arrange to do molecular analysis (specifically, 16s rDNA sequences) on at least the bacteria, if not the yeast as well. I’d really like to get good identification of these. Assuming they’re real Lambic organisms, they’re probably already in the databases somewhere and should be readily identifiable – assuming someone will let me use up some supplies.