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Dagl1

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Everything posted by Dagl1

  1. Dagl1

    Phage DNA

    I started explaining about viruses before I realised that phages specifically only talk about bacteriophages. I kept the information as you may find it useful or interesting anyway. The answer to your question, I would guess, is that it not great to do so for the phages. CRISPR systems may be one reason. Eukaryotic viruses (see below) can safely store their DNA within the nucleus, but this may be not feasible in bacteria. Instead even during latent periods they may only store their DNA within capsids. Sorry, I am not very familiar with phages;/ But based on this wikipedia article, I would guess that at least some viruses can stably integrate their DNA: https://en.wikipedia.org/wiki/Lysogenic_cycle This is in contrast to virus (please note I do not know enough about phages to say they do or don't have similar mechanisms) which don't immediately lyse their host cells. There are viruses which have a quiet phase, where they copy their RNA into DNA and store it close to the chromosomes within the nucleus. I am not very sure about the details/different types of latency, but some viruses can stay latent for super long periods of time. I think others can inhibit lysis for some time, thereby leading to larger amounts of viral particles. I am not sure if those that delay lysis (but do not become fully latent) also maintain viruses DNA as a stable molecule within the cell. I presume so, based on retroviruses existing, which insert copies into the chromosomal DNA of their host. https://en.wikipedia.org/wiki/Retrovirus Retroviruses such as HIV can also become fully latent, but recently I watched a video about Herpes virus (non retroviral) and how it escapes detection during latency:
  2. Dagl1

    Phage DNA

    Could you elaborate a little bit? Where have you heard/read this?
  3. Ahh at further inspection I spot my own mistake, I thought both sequences had the same amount of amino acids, and thus thought that in sequence 3 we would be looking at a frameshift mutation that would then mean we could puzzle our way through... Hmm I'll think of it, but with that extra amino acid it basically means anything goes (or well, I currently can't think of anything). Edit: the jump from serine to lys is also pretty strange if it would be a substitution reaction. I don't feel like going through it completely (so this is not per sé an answer, but a thing to explore), but I think an insertion at phe (sequence 3, position 4) could maybe be the answer, thereby leading to a frameshift and an additional amino acid. So knowing that the stop codons are UAA, UAG, and UGA. How would we go from Leu (position 4) to phe (position 4 in sequence 3). Leu = TTA, TTG, CTT, CTC, CTA, CTG. to phe (TTT, TTC) so that the the third nucleotide of what previously was Leu can combine together with Ser (TCT TCC TCA TCG AGT AGC) and the old stop codon (UAA, UAG, UGA) to form Lys (AAA AAG) and a new stop codon (UAA, UAG, UGA). This way X should (if this is the answer) not be too many different possible combinations. I am not sure if this will give you the answer, but its the only thing I can think of at the moment. Oh and of course assuming a simple substitution mutation for position 1. It may be also at the beginning, but that would make it even more complicated
  4. Could you explain what you have tried so far? What do they mean with sense-DNA sequence you think? Have you looked at sequences that code for these amino acids? I think you have to go and look at what happened to the sequence, maybe you can see which type of mutation happened (maybe name the types of mutations that exist).
  5. Washing probably is just putting the egg in the solution, then remove the solution, put another dose of solution, rinse and repeat x amount of times. https://www.sigmaaldrich.com/catalog/product/sigma/m7167?lang=en&region=NL&gclid=CjwKCAjwv4_1BRAhEiwAtMDLssbOtt671TSOSgmEJMC_4LWNIwG0EaaufWtwvujan2GZvopDMC6wRhoC3oUQAvD_BwE mineral oil: https://www.fertstert.org/article/S0015-0282(18)30177-8/fulltext Regarding the ratios, you may want to search for some methods in newer papers that use this technique, to find if newer methods use different ratios.
  6. This may help to answer some of your questions: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5828430/ One important thing seems to be the difference between fast and slow-absorbed protein. In the introduction they discuss some of the research regarding absorption rates, you could look at those to find an answer to 'how long it takes for it to restart and the body can absorb that amount again'. Other than that, many studies look at muscle growth, which isn't really the same as absorption (but I suppose they also look at urea in those studies)
  7. You linked to 1 study, that does not have a control group... Do you understand that that is no proof? Especially since the subjects were first told about the project, then had to say they were interested, thereby already producing placebo effects...
  8. Some points, what is an embryo? Embryonic development happens after fertilization. I presume you mean egg cell? Egg cells can be frozen, and are just in solution when a plasmid is added. Some transfection reagent can then be added (Fugene) together with the plasmid to transfect the cells: Eggs were exhaustively washed in M2 and then transferred to 50 µl microdrops of M2 containing the mixture of DNA and liposomes, under mineral oil. The plasmid used for the transfection was a commercial plasmid, pGeneGrip (GTS, CA) that encodes the green fluorescent protein (GFP) under the control of hCMV IE promoter/enhancer and is rhodamine labeled. For each experiment 125 ng of DNA in combination with 1 µl of a commercial liposome mixture (Fugene, BoehringerManheim) were resuspended in 100 µl of M2. Transfection was allowed to proceed for 3 hr at 37°C in an atmosphere of 5% CO2. Controls using Fugene only or the plasmid only were carried out. Sperm is collected, diluted into medium, and then added to the cells to fertilize them. Sperm were collected from the caudae epididymides of a vigorous stud male. Both caudae were punctured in 200 µl of pre-warmed capacitation medium (M16 medium supplemented with 40 mg/ml of bovine seroalbumin [BSA] and antibiotics) Sperm were diluted in capacitation medium to give a final concentration of 1–2 3 106 cells/ml. Capacitation took place for 3 hr at 37°C in an atmosphere of 5% CO2. At the end of the incubation, 100 µl drops from the sperm suspension were placed under mineral oil and the transfected eggs were transferred to these drops and left overnight.Twenty-two to twentyfour hours later the inseminated eggs were examined for signs of nuclear decondensation.... etc. Does that answer your questions? (note that I just found the first article that described this, nowadays there may be many different methods, but they probably follow the same base steps). From https://www.ncbi.nlm.nih.gov/pubmed/10862002 Carballada, Rosa, Tedla Degefa, and Pedro Esponda. "Transfection of mouse eggs and embryos using DNA combined to cationic liposomes." Molecular Reproduction and Development: Incorporating Gamete Research 56.3 (2000): 360-365.
  9. I think converting a healthy cell into one growing out of control, could be possible through epigenetic means. Highly increase transcriptional activity of growth-related genes, possibly decrease surveillance genes. Whether that would work (and not induce apoptosis or other forms of cell death), I am not sure about. But please note that tumors are generally mutating a lot. So even if you could start inducing a cell to become a tumor cell, being a tumor cells means mutating a lot. So at that point we are not really only within the epigenetic realm. Either way, I don't think we currently can do this, but I feel that in theory, creating uncontrolled growth through epigentic means is more feasible than turning tumor cells into non-tumor cells (but I suppose that uncontrolled growth is not exactly the same as being a tumor, so not sure if that really counts in your eyes).
  10. Probably not, epigenetic modifications can induce transcriptional changes, but if a tumor has a mutation in a gene necessary for its survival, then epigenetic modifications will not help to get around that mutation. Of course, you could alter some characteristics of the tumor (but it will probably be changing those back), but you cannot bring it back to an healthy condition without changing the genome as far as I can think of. How one would do that is even more difficult, you would have to induce methylation and histone modifications at many many sites, additonally you might want to change the location of the DNA within the nucleus to be closer or further away from the nuclear lamina. Some of these things you could do with very targeted dCas9 systems, which recruit factors. But then you would have to provide possibly hundred if not thousands of sgRNAs, and the question is if that would work. At that point it might be easier to 'just' try to find and edit away every mutation. Maybe other people can think of some ways that you can do this, but I myself can't come up with any way.
  11. Maybe you should answer questions, instead of referring back to people reading your article. Assume they have, and if they still have questions answer them, or direct them, with a lot of help and all the necessary steps, to the right answer.
  12. I was surprised to hear that Covid could target neurons, so I looked it up and found: https://www.sciencemag.org/news/2020/04/how-does-coronavirus-kill-clinicians-trace-ferocious-rampage-through-body-brain-toes Didn't fully read it, but may be interesting to some
  13. I am not very sure what the role of antibodies, or for that matter affimers, are in delivery of vaccines. I presume that affimers would work pretty well for Covid-19 testing. I don't know if antibodies will be completely replaced, but from what I have read (please note, I did not know about affimers before your post), they seem pretty promising in general, so maybe we will move away from antibodies. One thing is that most labs currently have large stocks of antibodies, and so even if everyone starts buying affimers now, we would still be using antibodies for a long time. But I did not evaluate the limitations, nor read deep enough into this to provide you with a real pro and cons analysis. Maybe other people on the forum know more or have better insights.
  14. They are smaller than antibodies, are generally more stable and can function in a larger pH range than antibodies. They are produced by phage display and several rounds can be completed in 12 days. They can be used for in vivo staining and can have more specificity than regular antibodies. They are also suitable for super-resolution microscopy, where regular antibodies are generally too large. They are based on a scaffold protein that is synthetic/modified, so that it only contains cysteine residues where we want them to be, thus allowing for more specific use of tags such as fluorphores and biotin. The scaffold protein itself can have additional attractive properties such as inherent protease inhibitor activity (although I suppose that activity is reduced when introducing variable regions, I am not sure enough about the biochemistry). I read https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4000234/ and https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5487212/ if you are interested into the details. The wikipedia has some additional answers but I presume you have read this already. https://en.wikipedia.org/wiki/Affimer. Hope this helps a little.
  15. Ahh I see, ye I didn't really consider the math differences when reading your post, apologies!
  16. There may be no oxygen? So what is it that we breath?
  17. Isn't it common practice to check for both normal distribution and equal variance, before applying tests to test if the nulhypothesis can be rejected? I remember using Levene's and Shapiro (and on the one occasions I had large sample size, kolmogorov-smirnov). Aren't they related in the sense that you need these assumptions for follow up tests (ANOVA's for instance)?
  18. Hi there, hope everyone is remaining safe and well! I was looking into phage-display library and have a question regarding the production. As far as I understand, one produces random mutations in some way (sources I am reading use degenerate primers), then these mutated primers are ligated into phagemids (not necessary, but this is I suppose safer as there is a need for helper phages), then these phagemids are transformed into bacteria. My question stems from the size of these libraries (being about 10^8 to 10^10), and the fact that each phage will contain the gene that encodes for that exact protein. So my question is, if we have our mixture of phagemids, each with different sequences, how do we make sure that each bacteria only contains one phagemid DNA particle (or at least, not several different ones). I imagine that if a bacteria contains two different phagemids, it could be possible that the phage displays a different protein than the DNA it contains. I thought, that we may just separately transform each bacteria, but due to the size of the library this seems unfeasible. I think my reasoning is going awry somewhere. Could anyone explain to me how we do this, or where the fault in my thinking lies? Sources I have used: https://www.jbc.org/content/273/34/21769.full.html pDN332 is a derivative of phagemid pHEN1 (7), in which the sequence between the NotI site and the amber codon preceding the gene III has been replaced by the following sequence, coding for the D3SD2-FLAG-His6 tag (22). Transformations into TG1 Escherichia coli strain were performed according to Marks et al. (6), and phages were prepared according to standard protocols (9). Five clones were selected at random and sequenced to check for the absence of pervasive contamination. The percentage of clones that express folded antibodies was determined by immunoblot and dot-blot analysis using anti-FLAG M2 antibody (Eastman Kodak Co.) and anti-mouse horseradish peroxidase immunoglobulins (A2554; Sigma, Buchs, Switzerland) as detecting reagents or protein A-horseradish peroxidase as described (9). Protein A binds strongly to folded VH domains derived from the DP47 segment (23). Reference 6 is https://www.sciencedirect.com/science/article/abs/pii/002228369190498U (behind paywall unfortunately, although here is a copy of what I think is the relevant bit): (d) Cloning of the XFI? gene repertoires Purified DNA of the scFv gene repertoires (1 to 4 pg) was digested with Not1 and either A’$1 or Xc01 restriction enzymes. (The 2 different restriction enzymes were tried in an attempt to increase the cloning efficiency.) After digestion, the fragments were extracted wibh phenol/ chloroform, and ligated into pHEN1 (Hoogenboom et al.. 1991) vector that had been digested with either &‘$I and lVotI or LVcoI and Not1 and electroeluted from a @So/o agarose gel (Sambrook et al.. 1990). Each scFv gene repertoire was combined in a ligation mixture which included 6 pg of digested vector. in a 100 /d ligation mix with 2000 units of phage T4 DNA ligase (New England Biolabs) overnight at room temperature. The ligation mix was purified by extraction with phenol and precipitation with ethanol. The ligated DNA was resuspended in 10 ~1 of water, and 2.5 ~1 samples were electroporated (Dower et al., 1988) into 50 pl Escherichia coli TGl (Gibson. 1984). Cells were grown in 1 ml of SOC (Sambrook et al., 1990) for 1 h and then plated on TYE (Miller. 1972) medium with 100 pg ampicillin/ml and 1% (w/v) glucose (TYEAMP-GLC), in 243 mm x 243 mm dishes (Nunc). Colonies were scraped off the plates into 10 ml of 2 x TY broth (Miller, 1972) containing 100 pg ampirillin/ml. 1:/b the first paper also refers to this paper (reference 9), although I don't think it is as relevant: https://www.embopress.org/doi/epdf/10.1002/j.1460-2075.1994.tb06308.x Thanks in advance -Dagl Edit: Watching a video of Creative Biolabs, they mention that the phage display library is produced from 10^8th independent E. coli transformations. How? I presume this is done with some machine, because I cannot imagine some poor souls transforming 10^8 different wells?
  19. 🤣 that took a turn to non-seriousness faster than I expected
  20. Is there a semipermeable membrane left in the paper towel? What is osmosis? What makes the water move, what is it based on? What is diffusion? (the rules of this part of the forum are not about straight up answers, and I am not sure if the fact that you are teaching junior high changes that)
  21. Dandelion... start with answering pzkpfw's simple case, that way you show you understand what people are trying to tell you regarding forces. Yes it has nothing directly to do with your example, but the principles remain similar... Most people here are trying to help you, but then you are saying that they don't understand what you are trying to do (which is fair, because you leave out a lot of information, but in this case, pzkpfw is just trying to help you out with some basic physics). We are 5 pages in, and I have yet to understand what exactly the point of this is, you are not trying to model reality, and you cannot invent new physics from things in an engine. But that isn't as important as just answering some basics physics questions, from the simpler case you can then apply those rules and logic to a more complex (your) example.
  22. I was thinking of free will in the sense of conditions (your actions now are the result of things that happened in the past, and the molecules in your body reacting to previous states, at no point do you have any 'say' in what the molecules do), not regarding the idea one could make some different choice based on reasoning, but I believe even with the definition that people can make meaningful choices, they are limited to whatever their personality/being/brainstate at that particular time allows, so that type of free will would be limited (but not non-existent). You confidently say what free will is, but I presume it is the definition (that I missed by not reading through the entire thread) you or people have agreed upon here, because I generally interpret free will in the sense as I described above. If we do follow my definition, I don't agree you would come into an endless loop when calculating everything, you would come to the conclusion that you calculating everything was part of what was/is going to happen, and it would all be part of some causal chain (of course, quantum mechanics and inherent randomness can make that chain deviate each time, but since humans (as far as I know) can't control the randomness of quantum mechanics, this deviation is still not in anyway influenced by some person). Regarding colour... well that is wavelengths of light being interpreted being picked upon our eyes right. Do electrons, protons, and neutrons not emit specific frequencies of light (even if they may not be within the visual spectrum)? (I honestly don't know, but always assumed they do). My point about the fact that calculating what YOU would do, would have to include myself into that equation, and to calculate what I would do, I would require my own environment, therefore me calculating whatever would be part of the causality, and therefore me telling you, and the specific reaction (you changing) would all be included in that calculation, thus you would have to change your calculation until the deviation would be so small that it is true. But I suppose it could also be an endless loop, depending if the act of knowing the calculations leads to divergent or convergent changes upon each subsequent calculation.
  23. I thought of it a little later as well;/ not really sure about it, but as a I mentioned, I didn't really think of it too much. I suppose each virus still utilises a specific mechanism, so maybe it would be possible to change the protein slightly so that it is only specific to Covid-19 binding, but to be honest, it sounds like A, a lot of hassle to go this route, B I am not even sure if that would work at all (and one would have to start testing other viruses that normally don't bind to this ACE-2 receptor either). The idea was interesting as long as it lasted hahaha
  24. Wait wait... you have no idea how to make the colour change based on there being virus or not...? And you want help with that part, yet you still call simple, just because sensitivity and specificity don't have to be super high? Do you have any ideas where to start. Just as a brainstorm (not to be taken too seriously, or at least it's not as simple, but) what about having ACE-2 receptors fused to an enzyme, which then changes the colour upon viral contact? Something akin to https://en.wikipedia.org/wiki/GCaMP. Not that that will be easy to just develop, but it came up in my head.
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