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[PSA] Biohacking FAQs #3, #4, #5
3. Gel electrophoresis uses ethidium bromide, which is a dangerous chemical. How are you disposing of it safely?
I'm not using ethidium bromide. There are a number of other gel stains which are much safer and easier to work with, such as SYBR-Green and SYBR-Safe. I use GR Safe, which is similar to SYBR stains but even better, because it can be stored at room temperature.
Per standard biosafety practices, I sterilize everything before I dispose of it.
4. Why is there toilet paper sitting on your lab table?
It's absorbent and good for wiping up spills, and it wastes less paper than using full paper towels to wipe up the occasional spill of less than 2mL of liquid. (The paper towels weren't in the frame. Nor was the sharps bin, or the fire extinguisher, or any other safety equipment. It's all within reach, though.)
5. Why are there Ziploc bags sitting on your lab table?
The bacteria I work with -- Lactobacillus acidophilus, Lactobacillus bulgaricus and Streptococcus thermophilus -- are what's called "facultative anaerobes": they prefer environments where there isn't much oxygen. (They'll grow when there's O2 around, but they won't grow as quickly.) So, when I plate them on a petri dish, I put the finished plate in a Ziploc bag. Then I put some vinegar and baking soda into an empty Coke bottle and capture the generated CO2 gas in a balloon, squirt the gas into the Ziploc bag, and close it up.
I asked a former boss of mine (a bioinformaticist whose PhD is in population genetics) whether he had any ideas for easy ways to provide an oxygen-free environment for my plates, and he said they used the same Ziploc bag trick when he was in grad school. It's ghetto, but hey, it works.
I'm not using ethidium bromide. There are a number of other gel stains which are much safer and easier to work with, such as SYBR-Green and SYBR-Safe. I use GR Safe, which is similar to SYBR stains but even better, because it can be stored at room temperature.
Per standard biosafety practices, I sterilize everything before I dispose of it.
4. Why is there toilet paper sitting on your lab table?
It's absorbent and good for wiping up spills, and it wastes less paper than using full paper towels to wipe up the occasional spill of less than 2mL of liquid. (The paper towels weren't in the frame. Nor was the sharps bin, or the fire extinguisher, or any other safety equipment. It's all within reach, though.)
5. Why are there Ziploc bags sitting on your lab table?
The bacteria I work with -- Lactobacillus acidophilus, Lactobacillus bulgaricus and Streptococcus thermophilus -- are what's called "facultative anaerobes": they prefer environments where there isn't much oxygen. (They'll grow when there's O2 around, but they won't grow as quickly.) So, when I plate them on a petri dish, I put the finished plate in a Ziploc bag. Then I put some vinegar and baking soda into an empty Coke bottle and capture the generated CO2 gas in a balloon, squirt the gas into the Ziploc bag, and close it up.
I asked a former boss of mine (a bioinformaticist whose PhD is in population genetics) whether he had any ideas for easy ways to provide an oxygen-free environment for my plates, and he said they used the same Ziploc bag trick when he was in grad school. It's ghetto, but hey, it works.
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Many of the DIYbio folks have biology degrees as well, I just happen not to.
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(Anonymous) 2008-12-30 03:40 am (UTC)(link)I think you are missing the point entirely here. As one of those "PhDs toiling in a university lab" as the AP article calls us, your response reads like you are asking for a formal grant proposal. While I just stumbled on this, I doubt such formal methodology has been laid out, and had this been proposed professionally I'd be aghast, but in this case, it's the JOURNEY that matters! Will the goal be achieved? Will the results hold up to our rigorous peer review process? Who cares! It's going to be adventure regardless. And that's the point.
Just reading the article and this blog certainly has given me new appreciation for basics like electrophoresis. I know I just grab my favorite rig out of my pile of assorted sizes and types, pour my gel, and hook up a nice regulated power supply without ever giving a second thought to how they were built and designed.
maradydd - keep up the great work. By its very nature, real science tends to attract both hecklers and legitimate concerned persons who worry about safety, animal rights, religion, politics, methodology, etc. Welcome to the club. You've done an excellent job not only on the science itself, but of professionally and rationally addressing public concern. I will try to make mention of your project as a way to help motivate my undergraduate students in my next course.
PS - I am and have been a regular IDT customer for many years.
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Don't let me spoil the science fun with actual, you know, science...
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(Anonymous) 2008-12-30 04:43 am (UTC)(link)As to how and if one can clone in their kitchen, I am certainly curious! MAKE magazine has already published plans for a homemade cycler, so if she can build a reliable electrophoresis rig, and put it all together with clean enough DNA preps, why couldn't she heat shock her vector into competent bugs? I should go make a username...
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Fundamentally I really am a software developer at heart, and a test-driven developer at that. I work by figuring out what I want a particular step of my work to do, figuring out how I'll verify that I've achieved what I wanted, then implementing and debugging till the tests succeed. It is a bit slow and plodding, but ultimately rewarding.
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What's your protocol?
For heat shock (which hasn't worked so far) I've tried a protocol from a patent that specifically focuses on lactic acid bacteria; it's a modification of an established protocol using PEG-8000, and I'll dig up the resource if you want to read it.
The heat shock protocol was pretty complicated, so recently I've been trying sonication, as described for E. coli here (http://nar.oxfordjournals.org/cgi/content/full/35/19/e129). No luck so far, though I'm going to do some more runs with better controls, and I'll try varying other aspects too -- so far I've only modified time, but I can also try temperature, CaCl2 concentration, adding some PEG, who knows; I need to do some more reviewing of the literature to get ideas, and have been keeping my eye out for a friendly microbiologist who works with Gram-positive bugs.
I would of course prefer to use electroporation, which was my old boss's suggestion, but electroporators are expensive. In addition to the wet-lab stuff I've been doing, I've also been working on a DIY electroporator; the logic side works, been having some problems getting the HV side to light up but that's what electrical-engineer friends are for. When I do it, I'll follow the protocol in "Optimization of technical conditions for the transformation of Lactobacillus acidophilus strains by electroporation", Kim, Han, Oh, You and Kim, J. Appl. Microbiology 2005.
Which plasmid/vector system?
At present, since I'm at the "get any old plasmid in there" stage, I have pGREEN plasmids from Carolina Biological.
Naturally, when we start trying to bang melamine deaminase in there, we'll need to build a custom plasmid for that. There are lactobacillus-specific cassettes out there; I didn't bring my lab notebook to Germany so I don't have the list with me, but I have a list of candidates. I plan to work with a supplier on the design of the eventual plasmid. We haven't decided whether to clone from an existing species ourselves, get someone else to do it, or have the gene synthesized from scratch, but I am leaning toward one of the latter two because I don't have the containment facilities to work with Pseudomonas or with Acidovorax avenae. (A. avenae is a plant pathogen, but it's a citrus pathogen and I live in California -- not inclined to take that risk.)
Where are you getting your primers?
When I do, it'll be from IDT. I don't think I'll get an ex-employee discount, but I love the company. :)
How will you choose your primers? Do you know how to design the construct and primers so that it will anneal successfully?
http://scitools.idtdna.com
Which restriction enzymes are you using?
Not at that stage yet. I plan to follow the lead of the BioBricks folks, though that will present its own interesting challenges, since BioBricks are designed for E. coli.
Are you using the shampoo/isopropyl alcohol method for DNA prep?
I've done it before, but the purity is kind of lousy. However, since I'm not at the point of cloning anything out of successfully transformed cells, that's another bridge to cross when I get to it.
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(Anonymous) 2008-12-30 11:14 pm (UTC)(link)Brian
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How will you quantitate your DNA?
Gel quantitation with Lambda/HindIII for starters, spectrophotometer later. I've got specs for a DIY spectrophotometer which is super simple to build.
Where will you get antibiotic for your plates?
The pGREEN plasmids confer ampicillin resistance, and I got my ampicillin in powder form from an aquarium supplier. I also have fish tetracycline, in case we decide to go with tetracycline resistance.
The questions about FRET, promoters, conformational changes &c are also further ahead than I am in my work -- we're still trying to figure out whether simply using GFP as a reporter (i.e., getting it to express when melamine deaminase is expressed) will do what we want, since I'm still working on figuring out whether melamine deaminase will be expressed all the time or if it will only be expressed in the presence of melamine. If it's the former, we happen to be working with a team at National Yang-Ming University in Taiwan which built a bacterial pH detector for this year's iGEM. If we go that route, we'll use the pH detector to detect the breakdown product of melamine deaminase + melamine (ammonia). For that matter, ammonia is very easy for the human nose to detect, so simply having the breakdown product might be enough. I don't mind trying an idea and finding out that it doesn't work; I learn as much from failure as I do from success.
If so, will you also be building your own SDS-PAGE setup and western blot apparatus?
Most likely, yes. I've held off on that because I haven't figured out how I want to handle polyacrylamide safely.
How will you be validating your results?
That's kind of a broad question, but of course repeatability, comparison to LC/MS and ELISA results, &c are all important. The NYMU guys are a big part of that stage of things.
Where will you get melamine to test with?
Aldrich sells it, 5g for $15.
Are you planning to make yogurt with the resultant bacteria?
Only insofar as it's a workable way to store cultures long-term when one doesn't have a proper freezer. I don't think it will be a very good delivery medium. Tablet form might actually be easiest, like the pills you can buy at the pharmacy.
Will you add antibiotic to the yogurt to maintain the plasmid?
Only if I'm using yogurt for storage purposes, and I sure as hell won't be eating it. I'm violently allergic to ampicillin.
Is the idea to ultimately mix food into the yogurt and look for fluorescence with the naked eye?
Or to culture bacteria on/in the food, yes. Being able to look for fluorescence with the naked eye (well, and a blue light) would certainly be optimal. Will we achieve that? Probably not without additional tweaking, since the concentration of melamine that is deemed dangerous is actually quite low. That said, there are some neat tricks in BioBricks for upregulating reporter genes.
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1. It looks like pGreen is a plasmid for bacterium mediated plant transformation, so not necessarily a great choice for cloning a gene into lactobacillus. It's been about 8.5 years since I was actively doing molecular biology, so I am no longer facile with all the cartridges and restriction sites, but it looks to me like pGreen is designed for gene transfer, not expression.
2. According to the web site, pGreen confers kanamycin resistance, not ampicillin, so if your plates are coming up empty (and in molecular biology, whenever anything goes wrong, the result is the same--empty plates), possibly it's because you are indiscriminately killing everything with ampicillin.
3. BUT, I did a very quick web search and it looks like lactobacillus is not necessarily susceptible to ampicillin. Ampicillin does work on gram positive bacteria, but not every gram positive bacteria is susceptible to every such antibiotic. Cloning is almost always done with e. coli, since the characteristics of the host cell are generally not of interest at the endpoint of the experiment.
4. If the protocol you found for transforming lactobacillus is complicated, then it is undoubtedly because simpler protocols don't work.
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In any case, the pGREEN I have certainly confers ampicillin resistance, as you can see in the diagram of the plasmid.
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Certainly, and that's why I haven't wasted my time trying a simpler heat shock protocol. The sonication work I've been doing is totally experimental, as I've found papers on sonication of yeast and papers on sonication of Gram-negative bacteria but nothing on sonication of Gram-positive bacteria. I'd think that if it were known not to work, someone would have said so already.
Regardless, electroporation of lactic acid bacteria is far simpler than heat shock, and it's what I'll be doing when I have the equipment.
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6. When you are talking about a cloned gene, expression is always controlled by the plasmid. The promoter on the plasmid controls gene expression, and it is almost always constitutive--always on. It will not "turn on" or "turn off" in response to the presence of melamine in the culture. In some biological systems, the substrate of a reaction will act as a promoter for a gene for a metabolic enzyme--but only sometimes, and only in the native organism. Once you clone the gene into another organism, there's not going to be a connection between substrate and gene expression.
7. Be careful when you heat agarose. It has a tendency to superheat and boil over. Add it to your buffer in a generously sized erlenmeyer flask, cover the flask loosely with saran, and microwave it for about 30 seconds at a time, then swirl and check to see if it's dissolved, using a heavy-duty heat-and-water proof glove (not an oven mitt).
8. What you are doing is basically 1970's technology. That is when molecular biology was just getting started and a lot of biochemists were kludging together equipment and supplies much as you are. If you check out methods manuals and seminal papers from the 1970's, you'll find pretty straightforward methods for building equipment and putting together apparatus. Even back in the early 90's, I can remember pouring my agarose gels into frames made out of tape. Acrylamide is extremely toxic. Handling it is not so bad, if you're smart enough not to eat it. But you will be generating toxic waste and you can't flush that down your drain. You will have to find an acceptable way to dispose of your toxic waste. Pouring PAGE gels is very tricky.
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I know. That's exactly what I've been doing. In fact, I've referred to papers as far back as 1954 (when the nutrient medium I'm using was designed -- and it's cheap and works great!) or even 1919 (a paper on making yeast extract).
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