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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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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.
(continued next comment)
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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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