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Cloning the gene for green fluorescent protein is fun. Lots of fun. Cloners have put the GFP gene into rabbits, plants, cats, fish, and worms, and made mutants that code for proteins in every color of the fluorescent rainbow. Teachers like GFP so much that every year, high school students throughout the U.S. clone GFP in biology class.

i-13e078415e7453be522ddebc09ed616f-tsien2.gif

Now, some people, who call themselves DIY biologists, have started cloning GFP for fun in their kitchens. Other people find this alarming.

From Yahoo news:

Jim Thomas of ETC Group, a biotechnology watchdog organization, warned that synthetic organisms in the hands of amateurs could escape and cause outbreaks of incurable diseases or unpredictable environmental damage.

“Once you move to people working in their garage or other informal location, there’s no safety process in place,” he said.

Jake Young, of Pure Pedantry dismisses this notion. He says that:

the more likely negative scenario is that these DIY labs will produce absolutely nothing.

and also that:

There is no greater risk of that than there is of transgenic bacteria escaping from any of the thousands of molecular biology laboratories in our nation. Genetically modified yeast and E. coli are not considered serious biological hazards because the modified strains require special growing conditions — including selection using antibiotics — to maintain the modification. Otherwise they revert to just plain old yeast and E. coli.

I agree with Jake that we’re unlikely to see amateur cloners causing environmental damage or causing outbreaks of incurable diseases. However, I do have a concern about home cloning.

For the record, if you read this blog, you may already know, I support DIY biology. I especially like the computer-based kind that I call “digital biology” and I have many posts on digital biology activities.

However, I’m also a microbiologist by training and I’m not comfortable with people engineering E. coli in their kitchens. I’m not worried about synthetic organisms, I’m worried about large numbers of bacteria with antibiotic resistance genes.

When students or researchers work with E. coli in a place like a school or lab, they have both the equipment to kill the bacteria afterwards, and someone around who knows that autoclaving your cultures is an important thing to do.

Your average kitchen, on the other hand, doesn’t have an autoclave, and or even a pressure cooker. I don’t know know if your average DIY biologist knows it’s a bad idea to pour E. coli down the sink.

Why would I worry about amateur cloners and antibiotic resistance genes?
i-38ae9a61d7a6f514839c61d2488e3a5d-selecting-clones.gifWe use antibiotic resistance genes in cloning because putting genes into bacteria is inefficient. Maybe only one cell out of a million might contain the gene you want to clone. So, we find that lucky cell by using antibiotics to kill all the other. Only the lucky cell with the antibiotic resistance “superpower” will live in the presence of antibiotics. All the other bacteria die.

Unless home cloners use proper safety techniques, like autoclaving or pressure cooking their cultures, they will be sending antibiotic resistant E. coli out into the environment at the end of their experiments.

Hopefully the DIY’ers take a microbiology lab course somewhere and buy a pressure cooker before they seriously get to work.

Comments

  1. #1 Jake Young
    December 29, 2008

    You make a good point that I hadn’t really considered. Hopefully, DIYers have enough training even as amateurs to remember to bleach their cultures before they pour them down the sink. And also you have to worry about playing around with E. coli in the same place you cook your food.

    I know that there are laws about these things for labs, but do you know whether they would apply to someone’s kitchen?

  2. #2 Sandra Porter
    December 29, 2008

    Thanks Jake,

    The only laws that I know of, that are even vaguely related, concern cooking and catering.
    You can’t cook something and sell it to someone without some kind of license.

    As far as I know, it’s perfectly legal to give food-poisoning to your family.

  3. #3 Tony P
    December 29, 2008

    I agree on the autoclave or pressure cooker.

    I was surprised though that the temperatures required are relatively low, on the order of 250F to 270F with pressures of 15PSI to 30PSI.

  4. #4 Sandra Porter
    December 29, 2008

    The temperatures are low relative to the temperatures you can get in your oven, but keep in mind, those temperatures are still higher than boiling water (100°C, 212°F).

    We can kill bacteria at lower temperatures when we use an autoclave or a pressure cooker because they generate steam and steam is more efficient at killing things because it’s better at denaturing proteins.

    Factors that prevent steam from penetrating also make the sterilization process take longer. Larger volumes, denser materials, stuff with more contamination, all require longer sterilization times.

    We can sterilize media at 121°C at 15 psi for 20 minutes. But to sterilize heavily contaminated materials, we autoclave things for longer, usually an hour or more. Biotech companies often use spore strips and put them in the middle of the load to check that everything really has been killed.

  5. #5 qetzal
    December 29, 2008

    To what extent would the std antibiotic resistance genes used for cloning really be an issue though? As I recall, genes like bla, kan, and tet were chosen in part because they encoded resistance to drugs that aren’t widely used clinically.

    And aside from the ab resistance genes, I’m not sure that putting E. coli down the drain is that big a deal. We do it every time we flush the toilet, after all.

  6. #6 Mariah
    December 30, 2008

    Ok, so let’s say Meredith gets her green melamine cultures going. Let’s say her neighbors want some. She gives them out, and the instructions to store them in the fridge (nice). They grow some up periodically to use. Meredith gives out the selection antibiotic, of course. Which they’ll all use properly (sure). All the neighbors now need instruction on technique and safety, of course. All now need pressure cookers/canners.

    I have a pressure canner. It isn’t big enough to hold a lot of equipment. And since these are heavy, not every stove can be used.

    http://www.allamericancanner.com/allamericanpressurecanner.htm

    Please note: All American Pressure Canners are NOT recommended for use on glass top/flat ranges because their weight may cause the glass/ceramic to break.

    Or everyone can get autoclaves. Sure.

  7. #7 Alan Dove
    December 30, 2008

    You must have worked in a much more stringent lab than I’ve ever encountered. The molecular biologists I know think nothing of dumping E. coli into the lab sink, whether it’s carrying resistance genes or not. Containment is nonexistent.

    That’s not to say they’re being careless. The standard laboratory strains of E. coli, and even most of the strains one might isolate from the wild, are generally not pathogenic. Remember, this is a bug found in virtually everyone’s intestines, and huge populations of it get flushed down toilets about a billion times a day. It’s a harmless microbe.

    We should certainly discourage DIY biologists from working with pathogens in their kitchens, but that doesn’t seem to be what’s happening here.

  8. #8 Comrade PhysioProf
    December 30, 2008

    To what extent would the std antibiotic resistance genes used for cloning really be an issue though? As I recall, genes like bla, kan, and tet were chosen in part because they encoded resistance to drugs that aren’t widely used clinically.

    That’s what I was thinking.

  9. #9 Becca
    December 30, 2008

    CPP and qetzal- well, there’s always acne and ear infections (for tetracycline and penicillin). Also, methinks non-penicillin beta lactams are very much used clinically; though I suppose you could argue about things like ear infection antibiotics and whether there’s any point given how widespread resistance already is.

    Alan Dove- from what I’ve seen, most molecular biologists will dump stuff down the sink w/o bleach. Most microbiologists (who may also be trained in molecular biology) won’t. Possibly that is because microbiologists are trained to know 1) E. coli, (sans shiga toxin) is somewhat harmless but 2) Bacteria love their horizontal gene transfer (see shiga toxin-on-a-plasmid, aka E. coli O157, aka “attack of the killer hamburgers”).

    I think DIY cloning is pretty cool. But I also don’t see any reason why you couldn’t get an autoclave put in your garage…

  10. #10 Sandra Porter
    December 30, 2008

    Okay group, I’m not very worried about E. coli K12, although, I think it’s irresponsible to dump cultures down the sink.

    Qetzal – you mentioned that we dispose of E. coli every time we defecate and flush the toilet. That’s absolutely right and that’s why we have water treatment plants. If you have doubts about the pathogenicity of human waste, do a Google search for cholera. Sure, cholera is caused by Vibrio cholerae and not E. coli, but it’s transmitted when water is contaminated by human waste.


    Alan – you observed that –

    The molecular biologists I know think nothing of dumping E. coli into the lab sink, whether it’s carrying resistance genes or not. Containment is nonexistent.

    I’m sure you’re right. I’ve also seen molecular biologists mouth pipette radioactively labeled materials, answer the phone wearing gloves (when working with radioactivity), and wear sandals in the lab. That kind of behavior is probably why the institutions where I worked made everyone take radiation safety courses.

    The fact that some people missed learning proper lab safety practices is not a good argument for everyone else to ignore them.


    Anyway – my concern isn’t E. coli K12. My concern is the spread of antibiotic resistance genes in the environment. Becca is correct. If there’s anything that we’ve learned from genomics it’s that microbes are very generous with their DNA.

    CPP and Qetzal – As far as the idea that those who developed vectors used drug markers that weren’t clinically relevant, I think that’s incredibly naive. It wouldn’t have been on people’s radar – and the NIH had extensive guidelines for working with recombinant DNA. Most people would have autoclaved their plates before throwing them away.

    I made some vectors myself, in the 80’s, and I simply used the tools that I had at hand. I happened to have a low copy number, broad host range, conjugative plasmid, with a tetracycline resistance gene on hand, so I used it as a starting point. I never looked in any medical journals to see if people used tet in the clinic. That wasn’t a routine practice.

    The markers that I’m familiar with – Kan, tet, bla, hyg, cat, gen – mostly came from transposons and ended up being used in vectors because they worked, they lacked common restriction sites and they were readily available.


    Are these antibiotics clinically relevant?

    Well, the kanamycin resistance gene codes for an enzyme called neomycin phosphotransferase. I have a tube of topical neomycin cream in my medicine cabinet. You can even buy it over the counter.

    Now, the ampicillin resistance gene, beta-lactamase – yes, people do use beta-lactam antibiotics. My kids have taken amoxicillin and ampicillin (Principen) for ear infections. There’s also carbenicillin (Geocillin) that people take for urinary tract infections

    Tetracyline is used, as Becca noted, for treating acne, as well as treating urinary tract infections, gonorrhea, chlamydia, Rocky Mountain spotted fever, pneuomina, and other diseases.

    I don’t think chloramphenicol is used very often, but it used to be used for urinary tract infections.

    So – no, you can’t assume that the antibiotic resistance genes in vectors are innocuous.

  11. #11 Tatiana Covington
    December 30, 2008

    The gene hackers are freeborn. Thus it’s for them to decide what they will do. Don’t like it? Lump it.

  12. #12 Sandra Porter
    December 30, 2008

    Tatiana –

    Grow up. When you work with chemicals or biological agents that have the potential to cause harm, it’s irresponsible to act like you have a right to ignore the impact that those agents could have on the environment or it’s inhabitants.

    In 2007, a UW professor, Dr. Daniel Storm, was convicted of a felony for dumping hazardous materials down the sink. (Seattle Times). Even a respected pharmacology professor can get in trouble for thinking that they’re “freeborn” and able to do whatever they like.

    It’s not unreasonable to ask DIYers to observe lab safety and have some consideration for the by products of their experiments.

  13. #13 qetzal
    December 30, 2008

    Dr. Porter,

    Yes, we have water treatment plants. So any coli that a DIYer puts down their sink ends up there, right?

    I don’t see how your comments about cholera are even slightly relevant here.

    Calling my comments about drug markers are “incredibly naive” is a big offensive. I’m virtually certain I have seen such comments in previous discussions on the potential dangers of rDNA.

    I, too, created my share of recombinant plasmids in the 80s, as part of my PhD work. I, too, used the markers that were available to me. However, I did look into issues of containment and inappropriate resistance transfer. That’s where I saw the discussions of which Ab resistance genes were least likely to present safety concerns. I wish I could find one of those references now, but I can’t.

    On a related note, experiments involving E. coli K12 that contains no conjugative plasmids and no transducing phage are officially exempt from NIH rDNA guidelines. BL1 is still recommended, but it’s only that – a recommendation.

    Finally, bla, kan, & tet genes are already incredibly common in the environment. The whole reason we found them and used them as markers is because they were present on mobile genetic elements, esp. conjugative plasmids like RK2, RP4, and many others. DIYers who use non-conjugative coli are very unlikely to have any significant qualitative or quantitative impact on the prevalence of these genes.

    If there’s any risk in DIY Bio, I think it’s in the slight chance that someone inadvertently clones somethink like a toxin gene, or mistakenly cultures some potentially dangerous microbe rather than coli.

  14. #14 Sandra Porter
    December 30, 2008

    No offense meant, qetzal.

    I agree that we used bla, kan, and tet markers because we had them.

    I don’t agree with the notion that the plasmid designers in the 70’s and 80’s specifically picked those markers after checking to find out whether those drugs were in clinical use.

    It sounds like you were an exception. But, I don’t think many of the people that I knew who were making vectors would have done this. In my graduate lab, we were more concerned about other features – like copy number, host range, and gene expression.

    Beta lactam antibiotics, neomycin, and tetracyclines were being used then and they are still are being used. We tell people not to dump their old prescriptions down the drain, why not be as cautious with the genes for resistance?

    As far as pathogenicity, I meant that E. coli isn’t always harmless and that’s why we treat waste water and ask people to wash their hands after using the restroom.

    There is something else, though, that you made think about. How do we know that the DIYers’ can identify E. coli when they see it? How do we know they won’t end up culturing MRSA or something else by mistake?

  15. #15 Alan Dove
    December 30, 2008

    Oh, come on. Let’s lay off hounding these motivated amateur biologists, and applaud their enthusiasm instead. These people like science so much they’re making it a major hobby. Hooray! Let’s have more like them, and encourage them to explore their interests further. We need more public interest in science, dammit, not less, and these folks are no threat to anyone.

    Antibiotic resistance genes are everywhere already. Putting a few billion more copies of bla et al. into the sewage system will have no effect on rates of antibiotic resistance in wild populations.

    Superbugs don’t come from making more copies of these already widespread plasmids – they come from applying some kind of selective pressure for bacteria to retain and maintain the plasmids. In other words, it’s not the plasmids in the sewer causing trouble, it’s the antibiotics being overprescribed in the doctors’ offices and ladled into farm animal feeds.

    How do we combat these much more serious problems? How about by encouraging more people to study and understand microbiology – even in their own kitchens? An educated public will have less tolerance for the policy gaps that really matter, and more understanding of how to use antibiotics appropriately.

  16. #16 TomJoe
    December 30, 2008

    If these folks can wrangle a -80 freezer, they should be able to wrangle up a pressure cooker. Anything short of that appears to be irresponsible. If you’re going to have a hobby, do it right. Sheesh.

  17. #17 Sandra Porter
    December 30, 2008

    Alan – I hardly think that asking people to dispose of bacterial cultures in a safe way qualifies as “hounding.”

    I think it’s great that people want to do science at home. I think it’s even better if it’s done safely, in a way that minimizes the risks to others.

  18. #18 Alan Dove
    December 30, 2008

    Sandra, I was responding more to the rising concentration of FUD in the comments than to your original post with the “hounding” remark. However, anyone advocating that amateurs acquire bulky, expensive, and potentially dangerous autoclaves just to delete a few harmless E. coli should be ready to defend that position a bit better.

    Sure, the pros use autoclaves, because we have easy access to them and sometimes work with bugs that require that level of caution, but even if we grant the dubious claim that an amateur could cook up something truly dangerous, much simpler methods will work.

    A 1:20 dilution of Clorox, a healthy dollop of betadine, or a few minutes in boiling water is more than sufficient to sanitize equipment. It won’t be surgically sterile, but neither is the inside of your refrigerator.

  19. #19 qetzal
    December 30, 2008

    Dr. Porter,

    First, an apology – I meant to say “bit offensive” not “big“. And thanks for your apology.

    Second, I think you’re right that bla, kan, and tet weren’t initially chosen based on any clinical considerations. Rather, I recall that clinical impact was considered during some discussions on which genes should continue to be used in routine cloning. Although again, this is from memory. I can’t locate any references now, so I could be mistaken.

    I agree that reasonable precaution is still appropriate. TomJoe has a good point that a DIY Bio club should be able to obtain a used autoclave at a small price. Anyone doing anything at home can still bleach their cultures & plates before disposing of them, though that’s a bit more tedious for plates.

    And yes, IMO, the biggest safety concern isn’t disposing of the coli or the Ab resistance genes. It’s the possibility that someone might decide to grow an inoculum from some random source, and generate a concentrated culture of some potentially dangerous microbe. I’m a molecular biologist, not a microbiologist, so I don’t really know how likely that is.

    What do you think? Assuming DIYer’s will almost always be using something like plain LB, how likely is it they could accidentally culture up something harmful from soil, or from some human source? (Hey, honey! Let’s see what kind of weird bacteria we can grow from Junior’s nose!)

  20. #20 Isis the Scientist
    December 30, 2008

    This is amazing. I remember being in high biology class and making karyotypes from black yarn. I read this thinking how amazingly far science has come and how fabulous it is that students have the opportunity to perform these types of experiments.

  21. #21 Sandra Porter
    December 30, 2008

    I have to say some interesting points have been raised here –

    – how serious is the threat from E. coli K12?

    – how much should we worry about releasing antibiotic resistance genes into the environment – when inside of E. coli K12?

    I find (usually after a bit reflection) that when I’m reacting automatically based on things that I learned 20 years ago – that questioning assumptions is good, and I even might be wrong or perhaps overcautious. I’ve been rereading the NIH Recombinant DNA guidelines and looking for papers on survival of E. coli K12 in the environment. I’ll write more tomorrow once I’ve had more to read them and digest.

  22. #22 Roland
    December 31, 2008

    How do we know some DIYer might not mistake MRSA for E.Coli?
    Seriously peeps, this is a distinction that would be discernible to well informed 6th graders.

    But really, dumping crap into the sink thats been tweaked,
    assuming it is safe based on some sense of
    well informed arrogance is
    unwise at best.

  23. #23 qetzal
    December 31, 2008

    Speaking of arrogance:

    How do we know some DIYer might not mistake MRSA for E.Coli?
    Seriously peeps, this is a distinction that would be discernible to well informed 6th graders.

    Sure, if said 6th graders have been informed how to make that distinction, and if MRSA vs coli were the only two possibilities. In reality, someone with little/no micro training might easily not realize that the colonies on their plates aren’t coli. And even if they do notice, they’re in no position to guess what the bugs are, or whether they’re potentially harmful.

    Now, if someone can knowledgably say that any potential contaminants are very unlikely to be harmful anyway, I’ll accept that.

  24. #24 Toaster
    January 2, 2009

    @Becca:

    NO! NO NO NO NO NO!

    Shiga toxin in enterohemorrhagic E. coli (EHEC) or any other Shiga-toxigenic E. coli (STEC) is NOT encoded on a plasmid! Shiga toxins 1 and 2 are both encoded on mobile lambdoid phage elements. Shiga toxin 1 is on H19-B and Shiga toxin 2 is on 933W. The prophages are integrated into the chromosomal DNA of the E. coli, not the plasmid.

    It is true, however, that EHEC does have a large plasmid, pO157. pO157 carries a lot of genes involved in pathogenesis, such as hemolysin and toxB, but it does not carry Shiga toxin.

    Sorry to get so worked up about this, but I’m currently working on figuring out how the Stx operon gets activated in vivo. It’s the knee-jerk scientist reaction of “Yes my Research IS relevant!”. No offense meant.

    On the DIYbio thing, though, I would be concerned about an amateur getting a hold of human pathogens, even something like EHEC, Pseudomonas, or Helicobacter. I would also be concerned about them using immortalized human cancer cell lines, like HeLa or Caco-2 because (from what I remember), both have the potential to be tumorigenic. Does the ATCC require institutional verification to buy something or can anyone order up some Bacteroides fragilis and T84 cells? If they want to use K12 or yeast or Vibrio fischerii or Dictyostellium, then by all means go ahead. I might even be willing to officially collaborate with them.

  25. #25 Toaster
    January 2, 2009

    @Becca:

    NO! NO NO NO NO NO!

    Shiga toxin in enterohemorrhagic E. coli (EHEC) or any other Shiga-toxigenic E. coli (STEC) is NOT encoded on a plasmid! Shiga toxins 1 and 2 are both encoded on mobile lambdoid phage elements. Shiga toxin 1 is on H19-B and Shiga toxin 2 is on 933W. The prophages are integrated into the chromosomal DNA of the E. coli, not the plasmid.

    It is true, however, that EHEC does have a large plasmid, pO157. pO157 carries a lot of genes involved in pathogenesis, such as hemolysin and toxB, but it does not carry Shiga toxin.

    Sorry to get so worked up about this, but I’m currently working on figuring out how the Stx operon gets activated in vivo. It’s the knee-jerk scientist reaction of “Yes my Research IS relevant!”. No offense meant.

    On the DIYbio thing, though, I would be concerned about an amateur getting a hold of human pathogens, even something like EHEC, Pseudomonas, or Helicobacter. I would also be concerned about them using immortalized human cancer cell lines, like HeLa or Caco-2 because (from what I remember), both have the potential to be tumorigenic. Does the ATCC require institutional verification to buy something or can anyone order up some Bacteroides fragilis and T84 cells? If they want to use K12 or yeast or Vibrio fischerii or Dictyostellium, then by all means go ahead. I might even be willing to officially collaborate with them.

  26. #26 Stacy
    January 6, 2009

    Hi folks,
    I’d expect science hobbyists to dispose of their waste properly the same way I’d expect any hobbyist to do so. Folks with dark rooms shouldn’t dump waste down the sink, same with paint or motor oil. I do understand the concept of not loosing antibiotic resistant E. coli into the environment, but honestly, a hobbyist works on a very small scale. Hot soapy water and a little bleach knocks the count down a lot. Compare that to, for example, a poultry farm or other animal facility where zillions of animals are given antibiotics on a routine basis. I’m pretty sure the E. coli-rich waste from those places isn’t autoclaved, or processed nearly as well as from a household. Basic safety of course, but keep it in reasonable perspective.

  27. #27 Sandra Porter
    January 6, 2009

    Stacy:

    Well, actually the motor oil thing isn’t a good example of environmental responsibility.

    Non-point source pollution from people changing their own motor oil is an environmental problem. We have some non-profit groups in our region who work hard to educate people about this.

    As far as photographers, and other kinds of hobbies, those differ in that the people who manufacture the chemicals expect them to be used by hobbyists and so they include warnings and information about proper disposal.

    With DIY biology, people are either making their own equipment and chemicals or buying them off of ebay. So, I don’t think they will necessarily know how to dispose of chemicals or biological materials properly. And, there won’t be any kind of information from manufacturers to guide them.

  28. #28 Stacy
    January 6, 2009

    Sandra,
    I agree completely. Since some people, sadly, dump motor oil down the drain, it is appropriate to educate them. It isn’t appropriate to make it illegal to own a wrench. I would much prefer to share a well with someone who tinkers with E. coli than with someone who fixes cars and dumps motor oil.
    Point taken, though, on the issue of people buying chemicals from Ebay and not knowing how to handle them or dispose of them – or not to mix certain things together a few inches from your face.
    I’m all for science collectives, for an “open studio” approach to biotech. Purchasing, storage, and disposal could be supervised, much like paint and solvents in an art collective. I wish there was one in the Maryland area; I’d happily volunteer.

  29. #29 Jonathan Cline
    January 6, 2009

    Hi Sandra,
    I’m the project lead on the melamine project mentioned by Meredith L. Patterson. You should note that she is using yeast for her experiments, specifically for it’s safety. The only other microbe we have considered using is e. coli ADP1, which is also known for it’s safety.

    I currently have not found a suitable test kit usable by “normal people” for melamine and it’s analogues as specified by the recent FDA limits. All the methods of testing for the dangerous chemicals require expensive lab equipment and university-level training.

    The FDA ex-chairman has noted during a CNN interview that less than 1% of all food imported/exported between the U.S. and foreign countries is checked by visual inspection. Less than half of that, he stated (0.5%), is ever inspected chemically. It is clear that “no one is watching the watchers” in this case. Even dioxin, which is a known dangerous chemical, was recently found in the U.K. food supply for farm animals, and tens of thousands of pigs (and others?) were slaughtered for safety. How is it that these dangerous chemicals are sneaking through testing, and contamination occurs so far up the food chain, onto our kitchen counters? It’s an unacceptable situation which I feel technology can solve. Giving more funding to the FDA and other agencies will not solve the problem, just as it doesn’t solve other similar issues (including health care). New innovation from outside typical channels is required.

    Although I don’t know what everyone in the DIY Bio group is doing, I do know that the projects I have discussed with others are no more dangerous than what high school students accomplish in an advanced placement high school biology lab. And also, safety is a constant ongoing discussion, including possibilities for safety training. Join the DIY Bio discussion group by searching google for the DIY Bio forums.

    I personally consider the melamine project to be an open source technology startup opportunity, with work distributed to motivated individuals like any skunkworks. We are all professionals in our respected fields. For example, I am an electrical engineer with many years of industry experience, and have now moved into biotech, as I feel the technology has reached a level of maturity which allows non-laboratory-biologists to make a very beneficial impact on the world.

    More details can be found on “openwetware” (search on google).

  30. #30 Sandra Porter
    January 6, 2009

    Thanks Jonathan and Stacy!

    Stacy, YES! I love the open studio approach! That would be my ideal way to do this.

    Jonathan, that sounds good. You might advise Meredith not to refer to yeast as “lactic acid bacteria.” At least, it appeared like that’s how she described them. I may have misinterpreted this.

    I do beg to differ with you on one point. There is a big difference between what the hobbyists do and the work in the high schools. That big difference is the presence of teachers. The high school teachers are required to learn and enforce lab safety and supervise the students. They also tend to order kits that come with instructions on safely disposing lab materials. The supply companies, Carolina, Bio-Rad, and others all treat lab safety as a big deal.

    I think it’s great that hobbyists want to participate in this. I attended the DIY bio meeting in Seattle and joined the DIY bio Google group. I’m happy to help advise and help keep hobbyists keep out of trouble.

    But I don’t assume that anyone will know what lab safety is without some kind of instruction.

  31. #31 Len Sassaman
    March 17, 2009

    Sandra — Jonathan meant “lactic acid bacteria”, or specifically lactobacillus acidophilus, and not yeast. In other words, we’re using yogurt bacteria, because of its safety. Meredith is fond of saying that lactobacillus is a good bug to work with, because the food industry does a large part of the quality control for you already.

    Meredith, a professional bioinformaticist, and I, a self-taught biochemist, have been tossing around the idea of “glowgurt” for years, between ourselves and amongst members of our intellectual community — one that includes quite a few PhDs, in everything from mycology to cell bio to population genetics to economics. We ironed out our protocol with these discussions, and convinced ourselves (and others) that glowgurt would work. We got a little silly, too — at one point, there was talk about freezing yogurt pops in plastic tubes and using them as edible rave toys. ;) I don’t think this is a “DIY”-specific thing, though; I’ve heard of grad students making glowing beer before, just for kicks. When people have reviewed our protocol, they don’t doubt that we can do it, or do it safely… they scratch their heads and wonder why anyone would want glowing yogurt. (I want to do an aequorin-based glowing yogurt, but GFP is the easiest fluorescent protein to find; photoproteins are harder to order off the Internet.)

    But, it just so happens that our work on making yogurt fluoresce fit nicely with Jonathan’s need for a safe, simple indicator mechanism. (This is basic black-box engineering techniques at work here, which is why it *doesn’t matter* if it’s yeast or bacteria as long as the host meets certain suitability requirements, safety and ease to work with being two of the most key.)

    The design is fundamentally quite simple. You can think of it simply as a state machine. We need the GFP to react in the presence of melamine or a predictable metabolite. So, we go find a gene for an enzyme that metabolizes melamine — it doesn’t matter into what, or how; just that it produces a unique signature of melamine metabolism — and we use some aspect of the metabolism process to trigger another biochemical event — essentially a switch, which activates a promoter we have tied to the GFP as our reporter. That’s your nano-engineering step. There was of course a lot of biochemistry we needed to work out to make this all fit together right, and we’re still going to need to determine the best melamine metabolism detector through experimentation, but fundamentally it’s just engineering. (And yeah, sometimes you mess up and purchase the wrong plasmids, or the wrong type of capacitor. A good engineer shrugs, makes a note of the gotcha, and moves on.)

    If you (or any other readers) would like to see the progress so far, or contribute your suggestions, please visit:

    http://www.openwetware.org/wiki/User:Jonathan_Cline/Notebook/Melaminometer/Brainstorming

    If I may make a suggestion, Sandra — try approaching us as equals. See what you can learn from us, in addition to helping “advise and help keep hobbyists keep out of trouble”. You’ll probably rub fewer people the wrong way, and heck, might even end up learning something too.

  32. #32 Sandra Porter
    March 17, 2009

    I could be wrong, but I’d think twice before buying those stories about glowing beer. It’s definitely possible to make beer from yeast that express GFP, I don’t doubt that, but there are other factors that make glowing beer somewhat impractical.

    For one, GFP isn’t a secreted protein and so, it’s likely to stay inside the yeast. Two, yeast are pretty heavy and tend to precipitate. As a consequence, three, unless you like beer with yeast on the bottom (I don’t), your beer probably won’t have much yeast in it and isn’t that likely to glow. Even then, you’ll most likely need a black light to see it.

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