CharonY

Usually it is not much of a problem. Generally speaking, if the phosphate concentration is in the range of roughly 10-100 µM it is not growth limiting anymore. For many cells very high P-concentrations can in fact be growth inhibiting. If phospate buffers are used in media, they tend to be in the in the lower mmol range to avoid this. So, even if phosphate is the sole buffer, usually the utilization by cells does not alter the its buffer capacity too much.

Maybe one should start off how antiobiotics (ABs) work. Most ABs inhibit protein synthesis or by binding at the ribsomes (the "proten factory" of the cell) but they can also effect specific enzymes. Given the fact that in these cases the function of the ABs requires effective binding to their targets, a possibility of resistance is to have a mutation that alters this binding sites, so that the AB does not bind anymore. These mutation can occur randomly and once ABs are present there is a selective pressure to maintain these mutations (provided they do not inhibit the primary functions of the ribsomes/enzymes, of course). This kind of resistance is generally not transmissible. However, target proteins may be protected against ABs by certain modification by other enzymes, which in turn can be propagated across a population via horizontal gene transfer (of which plasmids are one means). Other means of resistance include: -prevention of the entry of ABs into the cell: many ABs are transported into the cell actively. The cell does not do it on purpose, of course, but the ABs may be co-transported by transporters. Modification of these transporters may enable the bacterium not to import ABs -inactivation of ABs by modification or cleavage -export of the ABs: some bacteria possess export system that effectively pump out ABs so that the intracellular concentration remains low. Now regarding the evolution of plasmids: this is a very interesting topic but I think it deserves a thread on its own.

Nope.

Maybe we should start off with giving an overview what biofuels are instead of throwing in specific opinions to various sub-subjects of it? Just a thought. AFAIK any fuel produced in relatively short term by organisms is considered biofuel. This is supposedly to contrast it from fossil fuels which are also the product of organism, but which took a long time to form. To my knowledge there are basically four main directions: -ethanol -hydrogen -biogas (methane) -biodiesel (methyl esters) I am no expert on any of these fields (though I do have close colleagues in the areas of biogas and biodiesel formation), so take my thoughts with a big chunk of salt. One of the main problems to date is that all processes depend on microorganisms and that at this time point the whole process is still too expensive. Moreover, no single approach is likely sufficient to cover the need of fuels we have got today. At least not with further developments. According to some calculations for example, more is lost than gained in terms of greenhouse gas reduction and fussil fuel dependency when land is switched to crop growth for ethanol production (see e.g. Hedegaard et al., Environ. Sci. Technol., 2008, 42 (21), pp 7992–7999). Biogas is also now already used, especially in agricultural settings, but usually the output is just enough to power essential elements on the farm itself. However, they are usually truly powered by waste (in contrast to ethanol fermentation). A disadvantage is that methanogenesis is a somewhat complicated process that requires more than one species of bacteria to run efficiently. As such its productions is more difficult to control. Biodiesel is interesting as the one can engineer the organism simply to produce the required lipids and it does not require the rather inefficient means of fermentation for biomass increase. An interesting approach was to use either cyanobacteria or algae, which can directly produce the lipids required for biodiesel synthesis without the need to given them costly carbon sources (except CO2, of course). Still, this is to this date too expensive, though I expect much from this approach, as one is not dependent on growing other biomass first for fuel production.

Weeeelll even if the world does not end.. what is a PhD good for anyways

The advantage is not only on the side of the bacterium, but that of the mobile genetic element itself, too. From the viewpoint of the plasmid its fitness increases, if it can be persistent in as many bacteria as possible. As such it exhibits mechanisms that allows conjugation. It is the similar to, say, viruses. In contrast to viruses, however, plasmids do not exhibit a protein structure that enables them to infect cells. So, if the plasmids would actually decrease fitness of the cells (as viruses usually do), they would vanish, as there would be selective pressure on the side of the bacterium not to take them up, and the plasmid has no way of forced entry. As such generally only plasmids with contribute positively to bacterial fitness (e.g. resistance, metabolic or virulence plasmids), persist within a population. In fact, mobile genetic elements (including viruses, transposons, plasmids etc.) are the purest example of egoistic genes. Either force the cells to to propagate you, or make it worthwhile for the cell to do so (and at the same time introduce means of propagating yourself).

^ That is why I like to have biologists around that are not of the molecular flavour.

At least in Germany I have not seen it used either. At least not in normal educational settings. Maybe in assessment centers...

Hmm I am a biologist but I had a shortish collaboration with medicals. What I gathered with discussions with them (as well as with other people involved in research or rather work in clinical settings) is the following: - medical doctors get paid better. Simple as that. However, they usually have additional responsibilities (i.e. patients) -they tend to have less time for actual research. Between patients, administrative stuff (which is even worse in academia) and teaching there often is not enough time. Though to a lesser extent this may also be true if you are got a largish group in an academic setting. -respecte: well this is a double edged sword. As a non medical research scientist working in a clinic you often get looked own upon by the higher-ups (not on the assistant doc level, though as well as not in collaborations, as I found out). In the mainly medical community this may also be true, however, when it comes down to basic and often also applied research the medics take the back seat. Their training simply usually do not involve the necessary training to be full-fledged researchers. Some eventually learn it but then do less patient care, for instance. The quality difference is most evident by comparing PhD theses and the equivalents from medical students. So in research communities the medicals suddenly become junior partners. In many projects (as in that I was involved in) the role of the medics is often to provide samples for the researchers (apart from conduction clinical trials that is). Cutting edge lab work is thus rarely exclusively done by medical researchers. It simply boils down what aspects is more appealing to you. Basic research, applied research, using applied research or just collaborate on any level with others. Also think about clinical duties that medicals often got. Maybe you should ask Ecoli, though. I think he wants to make a dual degree.

If you are merely talking about logic, I wonder what you mean with reasonable. Because this : Implies to me that it is not necessary to be reasonable, as e.g. the existence of John's sister appears to be irrelevant. But here: You apply reason. You can formulate the theory that bodies do not need external forces to be moved, it is just not reasonable.

As I said before, most cell types survive outside the body for a certain amount of time, but most do not propagate. I remember that skin grafts, for example, could be kept viable for around 3 weeks (at around 4°C). There are of course always stem cells. I remember that the Fraunhofer institute is trying to create skin grafts based on ones own cells. I do not have the details, though. I think it was with the help of maybe adult stem cells. Most differentiated cells can not be kept alive or even growing and propagating for a prolonged time. That is why there is all the buzz about stem cells.

Wow, congrats. Do you have to wear silly costumes now?

I meant of course keeping them alive on a dish or flask. The activity of sperm cells degrades fairly quickly (though there slightly more robust than a number of other cells). You are aware that sperms used for fertilizations are kept frozen until needed? That is the whole point, isn't it? I would go for lymphocyte cultures. Even non-immortalized one can keep them alive for a few generations (far longer than three days). Mind you, the majority of cells would survive a couple of days using the right medium and incubator. What I am talking about are sustained cultures (as in established cell lines).

In theory yes. Though apparently there is rumored to be a list of good shops, where you get reimbursed fairly quickly whereas if you bought them from someplace else you may have to wait until you retire or get tenured, whatever happens first. I have the feeling that I shopped from one of the latter ones. I think that in truth the administration has just started the rumor so they can have some fun with the researchers, though. Over time I have accumulated quite a selection of cheapish stuff that I bought for various labs (including boxes, various tools, glass plates, PSUs and so on) now for which I did not got reimbursed while I was working at the respective institute. I could safe quite some money just by staying at one place for a little longer, methinks...

There is a difference between keeping cells alive and propagating them. Generally for continuous cultivation you have to use immortalized cell lines. These can be immoratlized tumor lines like the above mentioned HeLa cells, but they do not have to be. They generally have to be genetically altered, though (often with viral vectors). Just btw. it does not work well with sperm cells, as they are unable to propagate.

Well, I always wanted to be a research scientist, so a PhD was the only route for me to go. Now is it worth it? It really depends on what your goal is. If you want to be financially secure or have a family somewhat early, it may not be the best way to go. The reason is that you do not get well paid during your PhD time and the degree itself is merely the entry ticket, but by far not a guarantee for a position. If you get a job before a PhD, chances are good that during your working time your net earn may be higher than going the PhD route (at least in an academic career). If your goal is to score an industrial job, it may also be worth it, but it depends on the field, whether you really need a PhD. Some thoughts on an academic career: 3-5 years PhD time, where you get badly paid, 60-70 h per week workload advantages: you get a degree, the supervisor is interested in you getting the degree disadvantages: workload and pay after that: 2x2 years postdoc where you get little more money, but may have to change position after two years. Workload is same or more (as you get more involved in administrative and possibly teaching stuff) advantages:slightly higher pay disadvantages: same as PhD time, only that you are essentially a dispensable/migratory worker and chances are that the PI invest less funds in you. Depends on the group, though. This is the crunch time as in theory you are to build your career here, but as you are often totally dependent on which group you end up with, a wrong choice can stall your career (and you will have lost a lot of precious time). The postdoc time can be longer than four years, though. Getting a PhD position (and the PhD) as well as postdoc positions are comparably easy, as one essentially can be counted as cheap labour. Just by being persistent one is usually able to achieve both. The next steps are the tricky ones. ______________________________________________________ If you are lucky you may be able to get a faculty position after your postdoc. If you are very lucky it is a tenure track position (roughly 20% of postdocs manage to score a tenure eventually in the US). advantages: able to conduct research for a longer time in a row (usually ~5 years). eligibility for certain funding bodies and thus be able to establish a group. Much better pay than postdocs (often double or more). disadvantages: between group management, grant writing, teaching and faculty work there is usually no time for actual lab-wok. Thus one becomes dependent on getting a good group together, otherwise chances for getting new grants (and hence a tenure track or tenure position) diminishes.

Just wanted to add that normally only the initiation at the origin requires the RNA polymerase. The rest of the priming is conducted by the rifamycin resistant primase.

It is somewhat unlikely. The structures of pro- and eukaryotic cells (as well as transcription machinery) are so different that different mechanisms are required for viral infection and replication in each respective host. It is possible to artificially introduce phage material into eukaryotic hosts, though.

Well, I ended up buying equipment for my lab out of my pocket several times already because I did not want to spend 500$ on something that I could get for 30$ (+ some soldering).

IIRC = if I recall correctly And the paper was basically a proof of principle paper. It was possible to transfect somatic pig cells with a vector carrying a GFP gene and promoter and let the cell express it. Afterwards the nucleus was transplanted into denucleated oocytes. This precise setup is not likely to be useful, but it demonstrates that it is feasible. More useful experiments would include site-specific insertions (or fusions) so that expression can be tracked in vivo. Edit: too slow...

50 tons? Sorry cannot help you there. I only had research-sized fermenters (up to 50liters). Depending on setup they went for around 80-100k (small benchtops for around 20k). You probably should just ask for quotes from manufacturers available in your country...

OK let me try to clear it up a bit. The primary role of RNA-dependent DNA polymerase is the creation of RNAs, including mRNAs. Inhibition of the latter is the reason for the antibiotic effects of rifamycins. I have to admit that I am guilty of goading you a bit, as I was interested to see whether you really understood what you were saying, or whether you were just parroting. Let me point out where you went wrong: This is definitely wrong as the creation of DNA from RNA is the replication mode of mobile genetic element (wink) and not of the respective host organisms itself. This is kind of half right. The RNA polymerase itself is not involved in DNA replication, but their products are. The most obvious one is of course that no mRNA is synthesized, including those required for DNA, but also (arguably more important) for all other cell functions. Truth is, there is a link between RNA and DNA synthesis that you have not really mentioned, though. The papers hint at it, but they really dealt with replication of virus DNA. The papers actually identifying the link came much later (though there was already one in the 70s postulating a direct involvement, based on circumvential evidence). One of the initiation modes of DNA replication near OriC is mediated by a DNA-RNA hybrid (that initiate the unwinding) and IIRC the RNA portion is synthesized by the RNA polymerase. I was waiting for you to point that out (and I may have been slightly impressed if you did). But as I said, rifamycins inhibit all types of RNA synthesis mediated by the DNA-dependent RNA polymerase, not only those portions that pertain to DNA replication (otherwise you would have to categorize all antibiotics inhibiting protein synthesis also as DNA replciation inhibitors, as proteins are required for DNA synthesis...). And you think that is going to impress me or add anything to your argument? If you do not follow the content, showing off the cover will do nothing. And just btw OUP is a publisher as every other academic publisher the fact that it is a department of the university does not enhance its quality per se.And while I do not have the book I am pretty sure that if did describe rifamycins as inhibitor of DNA replication, it was due to the fact that it was used extensively for experiments regarding the DNA replication mechanism. But once more: it was mostly used as a transcriptional inhibitor, often together with chloramphenicol (with the above mentioned exception).

Shouldn't one ask the question before one is doing that?

I never said that imidazoles did not include antibiotical compouds. Only that not all imidazoles are antibiotics. Imidzaole is the the collective term, antibiotics are but a subgroup. I wanted to clarifiy (and other points). Your posts regarding rifamyicin only state what I said earlier. You do know what the function of the polymerase in question is, don't you (was there any point in posting them)? The question to the original post has been answered a while ago, though. Mobile genetic (retro-)elements did and do shape complex genomes. And there is more out there than that mentioned already. It is quite an interesting topic, though I would suggest opening up a new thread if you are interested in discussing it.

Actually it is a good point. However I am disregarding retrotransposons for the same reasons I am disregarding viruses: both are mobile genetic elements. Their mechanisms of replication ensure their own persistence rather than having a real role for the host. Or in other words, the reverse transcriptase does not fulfill host functions. In any case, while it is not my field of expertise I would not be surprised if these MGEs are likely precursors of viruses. Edi: I see what you mean now. Yes I should probaly have said RTs are a phenomenon of mobile genetic elements. Good catch.