Tr0x

This also came out recently: http://www.bbc.com/news/blogs-news-from-elsewhere-35323237 These little guys survived over 30 years at -20oC! I wonder if it had something to do with these "glass-like" proteins you're talking about. I also know that tardigrades can produce trehalose, a sugar which apparently preserves their cellular membranes when they're completely dried out. (original scientific article for those more curious: http://www.sciencedirect.com/science/article/pii/S0011224015300134) Makes me wonder about the interactions between these so-called glass-like proteins and the trehalose. Maybe having both mechanisms is what makes them so resistant to extreme temperatures?

Any basic neuroscience or possibly physiology text book would be a great resource. If by "nerve impulses/signals" do you mean the actual electrochemical process of relaying information through a neuron (i.e. action potentials)? Or, is it in a more general sense? Remembering back a few years to intro neuroscience: sensing pain and sensing light are very different processes, with a few similarities. As far as neurotransmitters from the eyes to the brain, they're pretty similar to any other signal. The neurotransmitter glutamate acts to pass on "positive" information (i.e. activation) while acetylcholine acts generally as an inhibitor, to pass on "negative" information (i.e. inhibition). The major difference with sensing light compared to pain, is that when light hits the retina, some nerves inside are actually inhibited which relays "negative" information, telling the brain the eye senses light (or to think of it another way, these neurons are active in the dark, telling the brain the eyes don't sense light!). Pain, however, is sensed through "positive" information, where nerves are activated (not inhibited) when you come into contact with something hurtful. (please be aware that this is a huge over-simplification, and there are many other neurotransmitters and processes at work!) Another difference between how pain is sensed, and light, is the structure of the neurons. I vaguely remember that there are several different types of pain sensing neurons, that act at different rates (i.e. so you feel an initial sharp pain, followed by a dull ache if an area is injured). The neurons involved in sensing light are also diverse, meaning there are many specialized types. Hopefully that gives you somewhere to start...again I'd recommend a neuroscience text book rather than info gathered from here!

Short answer The top layer, probably Long answer The supernatant is generally the top most layer, but it depends on what your desired final product is. My guess is that the "goopy" middle layer contains some clotting factors/platelets as you said this is from whole blood. This would probably not be the supernatant you're interested in if you're going for erythrocyte lysate. It all depends on your centrifugation protocol though, as the layers can mean various things.

Oh wow how could I forget that?! I need to brush up on my basic biology apparently The article I cited stated that the organism didn't have any significant membrane/cell wall beyond the ordinary. This suggests that the cytoplasm is likely acidic as well...but to what degree it didn't say.

The molecular components of cells are actually quite fragile, and require a protective coating of sorts to survive in extreme environments such as extreme pH (i.e. cell membranes/walls). Acidophiles usually have powerful ion pumps within the membranes, which effectively maintain a neutral pH in the cell. None of that really answers your question though...just showing that life can generally get around having to completely change its biochemistry in order to deal with acidic environments. A pH of 0 in the cell and nucleus would be tricky to withstand, as protein folds are very sensitive to pH. Assuming the pH remains constant, and given enough time, pH sensitive proteins could conceivably evolve to better fold at low pH (though they may not maintain the same efficiency or even function) . But at this low of a pH, hydrolysis would be running rampant, and probably chewing up peptides, and sugars (i.e. phosphate-deoxiribose backbones of DNA). If the organism could somehow shield it's DNA chemically (i.e. glycosylating it constantly) then MAYBE it could withstand having a pH 0 in the nucleus. But this would require a rediculous amount of energy for the cell...and converting the low pH into a form of energy wouldn't work in this case, as that would require a gradient/a higher pH in the cell than the outside. As for the lowest pH an organism can adapt to...well apparently you can ignore most of my above conjecture as there is an archaea that can survive pH 0...and apparently without a cell wall! Review articles citing the one I linked to would be worth a read. Whether or not the pH is acidic/neutral in the nucleus wasn't discussed. Oh, and while looking up answers for this, pH can apparently even be negative! Now THAT would be tricky to survive!

I'm just curious if anyone has worked with phage display before. It's a completely new technique to me, so I'd appreciate any insight you could provide on your own projects, or what you've heard/seen done with phage display. Specifically regarding phage types you've used, proteins you've worked with, and library construction/screening. I know it's been around for a while, and there are a variety of iterations of it, so discussion of any aspect of it is welcome. Opinions regarding the technique would also be interesting...especially your thoughts as to why it's fallen out of favour in recent years. A bit of background on my own project: - just starting work on applying phage display to my protein of interest - will begin with just expressing it, then expressing a novel protein similar to it - possibly moving onto a library to screen for mutants via biopanning against a target - my proteins are fairly large (>400 aa), so I'll be using this kit

Something you'll love doing! Personally, I love doing research, planning projects, and teaching so a professor is my dream job. Of course, it has to be in a field that I enjoy and working with decent people in and out of the lab.

This is the best place to start. Figure out what interests you, then try to match that to what the labs/faculty are researching at your institution. Don't be afraid to suggest new ideas to professors either. If you think you can but a different spin on what they're working on, pitch them your idea.

My guess is that your dNTPs are contaminated. You said that "The higher dNTP concentration, the higher intensity of the second band" which immediately suggests some type of contamination in them. dNTPs get contaminated fairly easily, as they're usually used by many people and for a variety of DNA work. dNTPs are also pretty cheap, so it should be easy to get a new batch. Also, be very careful when loading your gels. You said that bands are occasionally showing up in all your wells (I assume you mean ones without samples in them too). Since screens for DNA are generally really sensitive, they'll pick up even the smallest bit of spillage into other wells. A less likely reason for this would be different conformations of your DNA. Twists, hairpins or loops will travel through the gel differently, but there would have to be spillage to explain the bands appearing in the negative control lane.

I suggest you try reading the sequels in the Ender's Game series. They're more about morals, philosophy and the ethics of coming to terms with other intelligent life, all set in a really great sci-fi backdrop. There's not so much action/teenagers whining as there is in the original Ender's Game (which is still a great book in its own right). Too each their own I suppose. Getting back on track: I just finished "The Slap" by Christos Tsiolkas and am nearly through "Slaughter House Five" by Kurt Vonegut. "The Slap" was definitely out of the realm of my usual reading, but it`s a great treastise on modern life told through the eyes of six very different characters. I highly reccomend it.

Well a dichotomy is kinda inevitable since they're about completely different things: logic and reason vs faith and emotion. They're all human characteristics! So trying to discredit logic with emotion or faith with reason is impossible. Yeah, I'm always going to believe science is better, but I'm not going to go around lambasting other people's beliefs. And I do believe it is about education, as there are a LOT of misconceptions on both sides. Fundamentally however, we all just need to get along Getting back to abiogenesis...there are a lot of theories out there. My favourite is the "RNA world" hypothesis, as RNA can act as both an enzyme and as a storage device/replicator of information. The world before RNA is rather strange however...any thoughts?

Well you can't control what people are going to believe! It's rather silly that people just can't "live and let live", and that a disagreement over a question no one knows the answer to is now a "war". A better science education for everyone would help get the right ideas across. Of course creationists would argue we need a better religious education! Personally, I think creationism is dead wrong, and it's just going through a phase of revival (why? I have no clue! I think thats a question for another day!). That said, I don't care if people believe it. Whatever floats their boat.

I didn't watch the full video, but the first few minutes seem pretty standard stuff (if a bit silly with all the random stock photos). Abiogenesis is the leading theory in explaining how life came to be on Earth. I suggest you check out Dr. David Deamer as well, as he focuses on how protocells could have originated from rather simple chemicals in meteorites. It's cool stuff!

I'm a bit out of my element here (biochem grad), but I'd like to give my cousin some guidance regarding careers in theoretical physics. This is really to alleviate pressure from her parents, as they're skeptical she can come out of academia and apply her degree (she's nearly done her 1st undergrad year, so there's still a long road ahead). So I have a couple questions: 1) After her undergrad, what's a good next-step for a recent graduate of theoretical physics? 2) What sort of jobs/other fields can leverage a degree in theoretical physics? (And yes, I understand that asking advice on applying a degree based on theory is rather ironic!) 3) If you have a similar background, a breakdown of your own personal academic timeline would be very helpful! Thanks, -Tr0x

The best answer to this is that it was a "lucky streak". There's a 50% chance of having a female. So the probability of having 21 females and no males is... = 0.5^21 = 0.000000477 = 0.0000477% Or odds of 2096435:1 Given that there are many more than 2096435 mating cats in the world, I'd suspect this situation is not uncommon (but still pretty cool!) Another possible explanation for this would be if the father had Klinefelter's syndrome. This means he would be a "XXY" male, so instead of a 50% chance it would be a 66% chance in favour of female offspring, or odds of 624999:1 for this same situation occurring. I don't know if this can happen in cats, but it can happen in humans and mice apparently.

Immunoblots are generally the first step to confirm that your protein is physically "there" (i.e. in your sample/being produced by your system) Pros: Quick and relatively cheap (dependent on how expensive your Abs are) Quantifiable if compared against known concentrations of your protein of interest Cons: Doesn't give you any indication of activity, only showing the total amount of protein present Heavily dependent on the accuracy of your Abs Enzyme assay kits are useful if your protein of interest is known/relatively common/a substrate is known. Pros: Usually quick, and very reliable results on the activity of your protein productCons: Taylor-made to suit your protein product, meaning it has to have known activity (you can of course devise your own assay if you know a substrate) Can be more expensive, and usually requires additional equipment (i.e. plate reader) Only shows how much of the protein is active, and not the total amount produced These assays are generally complementary, and it's rare to see a paper without some form of both.

That's a good point. Having a strain that produces both isn't a necessity; merely a way to avoid having to purify so many possibly useful proteins by looking for interactions while the bacteria were still growing. I'll have to think on this before I decide which way to go. The concept of producing both will be tough to let go of, but the time involved will probably be the same either way.

That's a great point! However the assay is meant to screen a whole lot of them easily in order to find the mutant that works. So in order to purify each mutant proteinn from each of the producing colonies will take far too long. I'll probably end up doing that eventually but the first screen is "quick and dirty" in order to sort through all the colonies. Another question: does anyone know of an e coli strain off hand that will be suited to express two recombinant proteins at the same time? I've been using BL21 DE3 so far, and it's worked well for the one protein. The papers I've read regarding this have also used this strain.

Yes, in short it's just the isolated protein functions that I'm interested in. The proteins I'm trying to express are human, meaning they shouldn't have any effect on the e coli colonies, other than reducing their growth rate (which I've observed) as they're making a lot of extra proteins. As for secretion kinetics, I'm aware it's going to be less than ideal but I shouldn't need a whole lot of protein secret to observe it in the assay (fingers crossed that introduces enough). Thanks for the guidance so far! It's great to bounce ideas around. My impression then is that using standard techniques to create a plasmid with two secreting proteins instead of one should do the trick, albeit with a lower yield of each protein.

Yes, ideally I want to have a single strain secreting both proteins. The question I want to answer is whether I can get "protein 1" to inhibit "protein 2". In other words assessing the function of the proteins in vivo. I already have an assay working to detect functioning "protein 2", but the trick is to get the other one to express and secrete too in the same strain. If it would help, I could provide a bit more detail about the specific proteins, however I don't think it's necessary. Thanks for the help so far!

Hello forum! First time posting here but I intend to stick around. I'm wondering (and hoping) if anyone could provide some insight on expressing and secreting two independant proteins in E coli. I've scoured the literature and have found plenty on dual expression (see this article), but nothing that includes secretion too. The idea so far would be to use either a pre-made expression vector like Novagen's Duet and then adding signal sequences in the ORFs to secrete the proteins. Alternatively, I could design my own dual expression vector to do the same thing. I'm hoping to have this ready by September, although I realise it's pretty ambitous considering there's such little info on it. I already have one of the proteins secreting fairly well on its own, and am starting on the second. A stipulation is that I want to be able to continue using cells since I'l eventually be selecting for activity. So the cells must survive the procedure. Ideas?