text:
Purpose of dilution streaking or streak purification
In dilution streaking a drop of liquid culture is collected with an inoculating loop and then streaked across the agar plate surface. At the end of the streaking the number of organisms clinging to the loop until only single cells are deposited at a given location and you get colony-forming units.

I am wondering now what exactly the purpose of this streak purification is. If you want to grow your bacteria why would you not just put a few drops of the liquid culture on the agar plate? Why is it particularly useful to use this technique? Or, why would you choose this method over the "spread plate" technique, where a liquid culture undergoes a set of dilutions first and each dilution is then placed directly on the surface of separate agar plates.

The purpose of this technique is to dilute the bacterial cells so much that you get single cells, as can be seen on the image of the plate. You can then pick the colonies grown from these single cells, characterize them, test them for the mutations/plasmids, or whatever change you introduced, and can be sure that you have only the descendants of a single cell in your culture.
This way you ensure that you don't grow a mixture of different cells with different characteristics, of which one will eventually overgrow the other one. The streaking is a fast and reliable technique, which works especially well when you want to test a larger number of bacterial cultures. It also allows to check a stock culture very fast for contaminations with other bacteria by simply inspecting the single colonies by eye (different bacteria often look different).
Diluting the cultures would work as well, but needs to be done in more than one step and is more work when you want to do it for more than one culture. Also for dilutions you often plate more than one plate to be sure to have a plate with single colonies (when the cell number is unknown).

Maybe you were also implying this, but it's a fast method to see if your stock contains one strain or multiple (assuming you can distinguish them by eye).

And if you don't know how many cells you have you'd also use more than one plate when you plate dilutions.

@VonBeche Good points, I added them.

Great answer, I loved this technique as it is so quick to quickly get single-cells/mono-clones. One can not practically do serial dilutions when you have 10s of cultures or cell types to go through.


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What does the term 'glycogen mobilisation' mean?
I read that glycogen is a mobilised store of glucose:

Glycogen is a readily mobilized storage form of glucose. It is a very large, branched polymer of glucose residues (Figure 21.1) that can be broken down to yield glucose molecules when energy is needed. Most of the glucose residues in glycogen are linked by α-1,4-glycosidic bonds. [Source]

When looking online, mobilisation refers to something that is capable of movement.
I think glycogen mobilisation has something to do with glycogenolysis but not sure how to easily define glycogen mobilisation.
How would you define glycogen mobilisation in the easiest definition?

It would be helpful to see the context in which you encountered the term.

I think the key to understanding mobilization in this context is in the second sentence:

[Glycogen] can be broken down to yield glucose molecules when energy is needed.

The authors are using mobilize in a figurative sense to suggest that glycogen can be quickly and efficiently metabolized to suit the energy needs of the body, like an army may be mobilized to fight a war.  See definition #2 on Wiktionary:

mobilize (verb)
2. (transitive) To assemble troops and their equipment in a coordinated fashion so as to be ready for war.

I think that @acvill more or less gets it, but I wanted to add a direct usage in context that makes clear the meaning.
I found this as the top hit when I google "glycogen mobilization". It has a lot of information and it is not straightforward, but it makes clear that mobilization is the process of breaking down glycogen into glucose. For instance, one slide says:

Glycogen phosphorylase usespyridoxal phosphate (PLP) a derivative of pyridixine (vitamine B6) as a coenzyme. B6 is required for the mobilization of glucose from glycogen.

As far as I can tell, "glycogen mobilization" means the mobilization of glucose from glycogen, therefore it is a special case of glucose mobilization in which the source molecule is glycogen. For example:

Glucose is a negative regulator of liver phosphorylase. – Glucose is not mobilized when glucose is abundant.

Additionally, wikipedia says:

...debranching enzymes mobilize glucose reserves from glycogen deposits in the liver.

and

glycogen debranching enzymes function in glycogen breakdown and glucose mobilization. When phosphorylase has digested a glycogen branch down to four glucose residues, it will not remove further residues. Glycogen debranching enzymes assist phosphorylase, the primary enzyme involved in glycogen breakdown, in the mobilization of glycogen stores.

So glycogen mobilization == glucose mobilization, in the context of human biology. That is admittedly kind of confusing.

+1 This is a good clarification

Thank you @acvill

Thank you for clarifying @Maximilian Press

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How can I change the payload of a bacteriophage used to transform E. coli?
I was looking at bacteriophages and how they're used to transform E.coli. While the whole process of how a bacteriophage works makes sense theoretically, I wanted to know how one goes about changing the default payload of a bacteriophage with the payload of interest (engineered plasmid) in practice?
Clarification:
payload - Genetic material that the phage inserts into the bacteria
default payload - Whatever genetic material the phage inserts into the bacteria naturally
payload of interest - Engineered plasmid that I want to insert into the bacteria

I would recommend looking at some methods sections. Takara has a lengthy guide for using different kinds of viral transduction methods in mammalian cells, which is a little different but quite similar overall, that may have some helpful low-level information. Here is a review that may be helpful, focusing on engineering methods for bacteriophage, with particular relevance to infectious disease applications. They go ove

Can I ask why you want to use bacteriophage as opposed to electroporation or heat shock? I can't say I've ever heard of someone using phage to deliver DNA. I know I said it in my comment on your last post about electroporation, but it was more of a mental spitballing as to why lentivirus wouldn't work on E. Coli.

@yp66t89 I am actually familiar with some projects that were using phages to do rapid diagnostics type assays. I only made the connection once you mentioned retroviruses. But that got me curious about how you actually setup the phage payload in the first place.

I am currently using a 'phage integrase' system to insert my gene of interest into P. putida and it is definitely a seamless process with high efficiency so I highly recommend it!
As for your question, the method through which we integrate the gene of interest using the phage integrase system is simpler than you think. We do not change the payload of a bacteriophage. We simply electroporate with two plasmids, one that expresses the BxB1 integrase and another plasmid that has our gene of interest that we want to integrate into the genome of P. putida. This integration is highly efficient, and it operates through a serine recombinase that recognizes specific attP and attB sequences in the plasmid that contains my gene of interest and native genome of my engineered strain. This method is significantly more efficient for engineering certain strains (typically non-model organisms) for genome integration purposes.
References for additional information on the system for your review:
Here is a paper that describes how we use the phage integrase system to effectively edit the genome.
https://www.sciencedirect.com/science/article/pii/S2214030116300438
Here is the paper that discovered and characterized the mechanism this system operates through.
https://www.sciencedirect.com/science/article/pii/S1046202310003142?via%3Dihub

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Why does plasmid cloning efficiency go down with increased size of insert?
I am setting up a cloning experiment. Briefly, I ran PCR on a gene of interest, cloned it into a vector (pGemTeasy) and bulked it up in bacteria. I have been advised to limit the size of my fragment to <10kb.
Say I have two scenarios:
1 - Insert = 2kb
2 - Insert = 10kb
Assuming we use optimal ratios fragment/plasmid in both scenarios. Why does the efficiency go down?
Here is a ProMega tech note showing the phenomenon for my vector.

Related (not identical) question: https://www.researchgate.net/post/Strategy_to_clone_larger_inserts_2-6_kb_into_T_Vectors_pGEMR-Easy

This article may have the answer but I don't have time to go through it now: https://academic.oup.com/nar/article/27/3/910/2902439

I suspect, but don't have any proof, that it is the physical difficulty of getting both ends ligated to a single strand - think of it like this:
Your 10 kb is essentially a long string, ligate one end to your vector, now try to get the loose ends of that now ~13 kb string in proximity to the same. All in a tube with (assuming nanomolar concentrations) approx 1011 other molecules all trying to do the same. Chances are that the loose ends will encounter another molecule and end up ligating to those rather than the end that will result in a closed circular plasmid.
In contrast, with shorter molecules, having the insert and vector ligated at one end should constrain the number of potential interactions by relative proximity, making it more likely that the loose ends will interact and ligate to form the circular plasmid.
Now, I am sure that this isn't the complete answer, as there will be DNA topology, reaction conditions, etc. to take into account, but I think it is a good approximation of the problem.


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Does gene orientation relative to origin of replication matter on small plasmids?
The recent question about forward vs. reverse strand got me thinking about directionality conventions in synthetic biology.
As noted in the answer to that question, if we consider only DNA in isolation, the strands are symmetric and there is no reason to prefer one over the other. A great deal of work in synthetic biology, however, is done on small plasmids, and there is definitely a directional context for these systems: the origin of replication.
The origin of replication is typically separated from the insertion point in an engineered vector, but the replication process is directional and goes around the whole plasmid. I am thus thinking that there may in fact be a difference in behavior caused by the interaction of transcriptional activity and the replication process, especially for a high-copy plasmid, where replication is much more highly active.
Does anybody know if this is, indeed the case, and to what degree is it worth being concerned about?

I like this question, and I had a similar thought when reading that "Forward or Reverse Strand" post. I've since found a 2005 publication by Mirkin and Mirkin1, which investigates the interaction between the replication fork and transcription machinery in context of an E. coli plasmid.
An excerpt from the abstract:

Studying Escherichia coli plasmids, which carry constitutive or inducible promoters in different orientations relative to the replication origin, we show that the mutual orientation of the two processes determines their mode of interaction. Replication elongation appears not to be affected by transcription proceeding in the codirectional orientation. Head-on transcription, by contrast, leads to severe inhibition of the replication fork progression.

To break this down, replication in E. coli proceeds at approximately 20 times the rate of transcription.2,3 From this, there are two kinds of collisions possible.

"head-on" collisions where replication and transcription complexes proceeding in different directions meet
"co-directional" collisions where the faster replication complex catches up to the slower transcription complex proceeding in the same direction

Mirkin and Mirkin find that co-directional collisions have little effect on the procession of the DNA polymerase III holoenzyme, whereas head-on collisions
cause replication to stall.

One can clearly see that when transcription from the P7 promoter is codirectional with replication, replication was not affected (Fig.3A). Head-on transcription, in contrast, imposed severe constraints on the replication fork progression (Fig.3B).

Altogether, it is a gem of a paper, and I suggest reading the full discussion to best understand the limitations of their experiments and their caveats in interpreting the results. (e.g. the different rates of procession of DNA Pol I and III, and the frequency of polymerase switching.)

References

Mirkin EV, Mirkin SM. Mechanisms of transcription-replication collisions in bacteria. Mol Cell Biol. 2005 Feb;25(3):888-95.
Gotta SL, Miller OL Jr, French SL. rRNA transcription rate in Escherichia coli. J Bacteriol. 1991 Oct;173(20):6647-9.
Hirose S, Hiraga S, Okazaki T. Initiation site of deoxyribonucleotide polymerization at the replication origin of the Escherichia coli chromosome. Mol Gen Genet. 1983;189(3):422-31.

That's a fantastic find!

Nice paper! Strange though that it doesn't mention how such collisions affect plasmid copy number and gene expression levels (quantitaviely speaking). These would be relatively easy things to measure, especially compared to the assays they did do.


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Conservation Law in Gene Regulatory Network modelling
I was going through the GRN modelling from Chemical and enzyme kinetics
by D. Gonze & M. Kaufman (PDF). The gene has 2 sites for activator/repressor. It say the DNA $D_0$ combines with activator/repressor at one of the sites leading to $D_1$ and then $D_1$ combines with $X$ again leading to $D_2$. It says then then total DNA is conserved in the reaction leading to $D_{Total} = D_0 + 2D_1 + D_2$. I want to ask why is there "2" with $D_1$ shouldn't it be $D_{Total} = D_0 + D_1 + D_2$. What I am talking about is given on page 49 of the document.
$$D_0 + X \leftrightarrow D_1$$ $$D_1 + X \leftrightarrow D_2$$
For first chemical reaction the rates are $k_1$ and $k_{-1}$. For the second reaction the chemical reaction rates are $\alpha k_1$ and $k_{-1}$.
Also, what will be the rate of the reaction for $D_2+X\rightarrow D_3$ when the DNA has 3 sites instead of 2.


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Can stand-alone DNA (no nucleus, no membranes, organelles...etc.) create cells?
I have been searching for an answer to the following question as part of my research but can't find a direct/complete answer yet.
Can stand-alone DNA (no nucleus, no membranes, organelles...etc.) create cells?
From what I have learned about the biology of the cell, it seems to me that it is virtually impossible, but I will like an answer from an expert on the subject matter.
Regards,
Alfredo Archilla

Too much information is missing. E.g. what is the DNA standing alone in (water? Saline? Primordial soup? Eukaryotic cytoplasm minus organelles?) Can you define "cell"? If you're asking if DNA can do anything by itself, it can't. It's just a language, a code, a blueprint, a complex set of instructions. A blueprint can't build a building. DNA can't build a cell.

Welcome to SE Biology. Please finish reading the [Tour] (we know you have not) and then the Help on asking questions. You will see that you are expected to do some research and thinking of your own before posting a question and demonstrate it in the question. What have you learned about biology that makes you think it is impossible? If you are correct, this does not make a question of interest to biologists, but more the use of this site to check whether you have the correct answer to some assignment. If so, this is not what the site is for.

DNA absolutely not, RNA maybe...
DNA can't even replicate itself without help
RNA on the other hand makes almost everything. It makes proteins, it makes DNA and we know it can at least bind and alter phospholipids. It can also make more of itself.
keep in mind the simplest living cells (prokaryotes) do not have nuclei nor most of the things we call organelles either.

https://www.ncbi.nlm.nih.gov/books/NBK26876/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC17937/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7122455/

Ribosomes are necessary for protein synthesis. Without organelles, RNA is basically useless, depending on your definition of "cell". Without membranes, what does RNA's ability to alter membrane permeability matter? Without mitochondria, there's really no energy for the cell (again, depending on what you consider a "cell".) I think this answer is incorrect, but that's just an opinion. The OP has not given us enough useful information, but as stated, RNA can't make a cell.

RNA doesn't make proteins. Ribosomes (which include proteins) and tRNA-complexed amino acids make proteins. You need chaperones to help fold proteins, post-translational modifications need to be made for proteins to be active, and they need to be localized correctly. You also need a supply of lipids and phospholipids to build membranes. All of this requires pre-existing "stuff" that is not RNA. If your answer was correct, I could leave my total RNA extraction sitting around long enough and cells would form in it. Obviously, that doesn't happen.

@MattDMo all the functional parts and the bulk of the molecules of ribosomes are made of RNA, the proteins are only some stabilizers. tRNA is amazingly also made of RNA. you absolutely do NOT need chaperones to make proteins fold the right way. chaperones make it more efficient they are not necessary. one way you can tell is viruses often have no chaperones  or post translational modification for their proteins.  lastly make is not the same as made of, I never even tried to claim membranes were made of RNA.

@anongoodnurse most cells do not have mitochondria, mitochondria is only in eukaryotic cells. please learn what actually makes up the simplest cells. also ribosomes are made of RNA. Proteins synthesis in its simplest form is rRNA reading mRNA using tRNA carrying amino acids to make proteins.

Is a ribosome an organelle? Yes, it is (OP stated "no organelles".) Can a ribosome assemble itself from RNA? No, it cannot. Please don't tell me what to learn; I've had more schooling than most (Physician, former Molecular Biologist). Your answer is simply wrong. Surprising, given your profile claims, that you can't see the errors in your answer. Besides, you spent your answer on what the OP's didn't actually ask. Cells have cell membranes. @MattDMo is correct, as am I.

@anongoodnurse can a ribosome assemble itself, yes, https://pubs.acs.org/doi/abs/10.1021/ar2000474 I note no mention of mitochondria, do you still think they are necessary? If you define a cell as having a membrane sure it has a membrane could something without a membrane cells have membrane replicate, yes, could it make a membrane quite possibly.  you seem to be confused about what the OP asks, it is not is something without organelles IS a cell, it is could it make a cell.

I'm not going to argue anymore after this. Even "simple" ribosomal formation requires the proper preexisting ribosomal proteins, a complete buttload of enzymes, rRNA, a cell-membrane dependent protein localization, etc. I'm not confused about what the OP asked. It's clear: "Can stand-alone DNA (no nucleus, no membranes, organelles...etc.) create cells?"


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What happens when you tag a protease with its own degradation tag?
I've been learning about the ClipXP, ClipAP, and Lon proteases. They are proteases from the AAA+ family, which seek out proteins tagged with certain peptide sequences, unfold them, and chop them up.
But what happens when you tag a protease with its own degradation tag? For example, what if you took DNA encoding mf-Lon and added its ssrA tag to the end, and then expressed this sequence in bacteria?
Would this result in any interesting dynamics? Will the protease start trying to degrade itself like an Ouroboros? Will it get stuck trying to do so? Will we observe any interesting dynamics when we incorporate this into a gene circuit?

The proteases would presumably degrade each other.

Yeah @canadianer. But I wonder if anything would be different from normal protein degradation. Maybe the protease concentration may have a lower and perhaps more stable steady state? Perhaps because the protease is evolved to degrade other protein, it might be more tightly folded and hence harder to degrade? Might it even get stuck trying to do so? what if two proteases bite each other 69 style?

You may be dealing with an example of autocatalysis and this may give rise to an S-shaped progress curve (that is, an S-shaped velocity-vs-time plot; not to be confused with an S-shaped velocity-vs-substrate plot). A very good example is trypsinogen/trypsin.  See Fig 2 of  ISOLATION FROM BEEF PANCREAS OF CRYSTALLINE TRYPSINOGEN ... by Kunitz and Northrop (pdf is free).  The kinetics of such a system is well treated by Frost and Pearson, p19

The process of self-degradation of a protease is called autolysis, and the rate of autolysis depends on many factors including temperature, pH, and enzyme concentration.1,2 While most proteases show reduced proteolytic activity with increased autolysis, some proteases demonstrate increased activity after they are self-hydrolyzed. The most notable example is trypsinogen, which, when cleaved initially by enteropeptidase, yields trypsin, an important digestive enzyme. Trypsin can then hydrolyze more trypsinogen molecules in a process known as autocatalysis / autoactivation.3

References

Chen XL, Shun CY, Zhang YZ, Gao PJ. Rapid monitoring of autolysis process of proteases by capillary electrophoresis. Biotechnol Lett. 2003 Oct;25(20):1763-7.
Khalil ME, Metwalli SM, El-Sebaiy LM. Factors affecting the autolysis rate of crude preparations of proteolytic enzymes from Bouri fish (Mugil cephalus). Food Chem. 1987, 24, 127-135.
Mayer J, Rau B, Schoenberg MH, Beger HG. Mechanism and role of trypsinogen activation in acute pancreatitis. Hepatogastroenterology. 1999 Sep-Oct;46(29):2757-63.

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Do spike-protein-based vaccines undermine the DNA repair system?
I ain't no biologist, but I came across a paper recently and tried to understand it:
SARS–CoV–2 Spike Impairs DNA Damage Repair and Inhibits V(D)J Recombination In Vitro
My question: Is it a correct takeaway that vaccines that are based on the full-length spike-protein such as the one from BioNTech may possibly be undermining our DNA repair system, rendering our immune system more vulnerable in general than it was before?

For those interested, chemist and blogger Derek Lowe has published a post on the article in question.

Note: in the time since writing my answer, the paper in question has been retracted. Here's the text of the retraction notice provided by MDPI in collaboration with the authors:

The published article has been retracted. Following publication, the first author contacted the editorial office regarding an improper experimental design with the potential to significantly affect the integrity of the resultant experimental data.
Adhering to our complaint procedure, an investigation was conducted. Both the chosen construct of the spike plasmid that contained a C-terminal fused with 6xHis tag and use of a GFP reporter system under overexpression conditions in the protocol were identified as having the potential to introduce significant ambiguity regarding the nature of the reported observations. The reliability of the results and conclusions presented have therefore been undermined. Furthermore, statements regarding the effect of the spike protein on the adaptive immunity are misleading as in this article no experiments related to the adaptive immunity were performed, and the full-length spike-based vaccine was not studied. Therefore, conclusions related to vaccine safety are not validated and lacked experimental support. This article is retracted and shall be marked accordingly. This retraction was approved by the Editor-in-Chief of the journal Viruses.

While the linked paper presents interesting results, the conclusions must be interpreted in context of their in vitro methods and cannot be readily extrapolated to understand the in vivo effect of SARS-CoV-2 mRNA-LNP vaccines. Here is their important caveat:

Although no evidence has been published that SARS–CoV–2 can infect thymocytes or bone marrow lymphoid cells, our in vitro V(D)J reporter assay shows that the spike protein intensely impeded V(D)J recombination.

They show that spike protein expressed in human embryonic kidney cells (HEK293) can localize to the nucleus and inhibit non-homologous end-joining (NHEJ), which is a vital process in the generation of diversity in the antigen-binding regions of antibodies. However, the way the human adaptive immune system works is such that the cells responsible for generating antibody diversity never internalize the antigenic protein. Once injected, the mRNA-containing lipid nanoparticles are endocytosed by dendritic cells. The mRNA is then translated and the spike protein is presented to T- and B-cells in lymphoid tissues to elicit an immune response. These T- and B-cells are the cells that carry out V(D)J recombination, but they are not the cells that are engulfing the nanoparticle load.
Two good reviews that cover this process in-depth:

Teijaro JR, Farber DL. COVID-19 vaccines: modes of immune activation and future challenges. Nat Rev Immunol. 2021 Apr;21(4):195-197.
Angeli F, Spanevello A, Reboldi G, Visca D, Verdecchia P. SARS-CoV-2 vaccines: Lights and shadows. Eur J Intern Med. 2021 Jun;88:1-8.

The authors explicitly suggest the first part of your question in their discussion section:

indicating that full–length spike–based vaccines may inhibit the recombination of V(D)J in B cells,

However, for this to have a meaningful effect on the immune system as a whole requires that a significant portion of B cells uptake the vaccine and express spike protein, which isn't demonstrated. The paper also, frustratingly, doesn't indicate if their results showing an effect of spike protein on DNA-repair occur in the presence of the non-structural proteins Nsp1, 5, etc., which as they mentioned are known to transport themselves to the nucleus. If spike protein can't get to the nucleus without these other viral proteins which are not included in the vaccine, then there's no reason to suspect the vaccine-produced spikes would have this effect.
With regards to the second part, "rendering our immune system more vulnerable in general than it was before", the spike-based vaccines have been demonstrated to generate a robust B-cell response, so we can rule that out. The immune functions full-length spike protein is suggested to impair are measurably functioning well in vaccinated individuals. In the cytoplasm of cells, mRNA of vaccines and the spike protein they produce can be expected to be recycled after a day or two, so long term effects of this nature are very unlikely.


text:
At what point in evolution is a new species identified?
As we know, a change in allelic frequency leads to evolution, and as these changes accumulate a new species is created.
My question has two parts -

A classical definition of species which is now not accepted everywhere:
Two different species are the populations that has lost the ability to interbreed among themselves to produce viable offspring.  I have read some of the answers on stack exchange addressing the genetic reasons for hybrid sterility. They frame faulty pairing in meiosis as the main reason, but if the genes are on the same loci in two different closely related species how does a change in allelic frequency lead to faulty meiotic pairing in the germ cells of the hybrid?

In the divergence of two populations sharing a common ancestor, allelic  frequencies are changing, but at what point are the populations called two different species?

Does this answer your question? How is genetic speciation defined? To quote one of those answers: "It turned out that the main differences between the two genomes were clustered around regions where chromosomes were rearranged. Although indicative of the cause of speciation, it can nonetheless also be the result of it. We can never be sure." Chromosome rearrangements are of course one of the most obvious mechanisms for bad meiotic pairing and non-disjunction.

what do you mean by chromosome rearrangement can you define and elaborate the process by which it happens?

ok this answers the 2 nd part of my question that when can we say two population are no longer the same species (:In other words, when two suspected sub-species are not able to produce viable offspring, they can be considered to be two species." but i was looking into the biochemical or genetic reason and their relation with changes in allelic frequency, though its a good answer.

Chromosome rearrangement: when structure of chromosomes changes. This is a very large and complex field of inquiry, and there are many mechanisms by which it happens. That is a large question in itself, I recommend doing background research and possibly asking a separate question if the term is unfamiliar. 2. Biochemical/genetic reason for speciation: I think that the take-home message is the one in the linked answer, which is that there is no single mechanism by which species are defined. I would consult the linked answer for more information about how species are defined.

Directly to your interest in allele frequencies: I don't think that changes in allele frequencies are going to tell you much about speciation. I think that they are an inexact and rather generic description of an extremely complex and context-specific process.

Please take the [tour] and then consult the [help] starting with [ask] questions effectively on this site. From this you will see that we expect questions to be focused (including one question per post), well-researched (in particular, you need to look for overlapping questions on this site), and clear. — In one sentence  you wrote "if the genes are on the same loci", which doesn't seem to mean anything, and "how does a change in allelic frequency lead to faulty meiotic pairing", which assumes something that isn't true. I strongly encourage you to do some background reading/research. ...

... I have found Khan Academy to be very helpful for learning the basics of a subject. You can then move on to Understanding Evolution from the University of California, Berkeley. After that there are many free textbooks available at the NCBI Bookshelf and many other online resources.


text:
For a biological AND Gate to work, do the two inputs need to arrive at the same time?
I understand that AND gates require the two inputs to be present for the output to be produced. However, if one input arrives faster than the other input does the output still get produced?  For example, consider the split T7 RNAP Expression System, where one input produces the T7 N-term and the second input produces the T7 C-term and both join to produce the output. So both need to be present to produce the output. If input 1 occurred first than the second input, will the output be 0 or can the N-term wait until the C-term is produced.
Thanks

It depends on what physical/biologic/informatic realization of a gate you are talking about. I suggest that you explain more clearly what you are talking about - as is, there is too much hidden information (known only to you) in your question.

It sounds like you are thinking about this like a programmer rather than a hardware or wetware engineer.

An AND instruction in a computer program is evaluated at a particular time, and only the values at that instant matter. Thus, the concept of a value "arriving" can makes sense and one would need to worry about times of arrival.
An AND gate (whether biological, electrical, or other) operates continuously, and is always producing a signal based on what its input levels were in the recent past, with the time delay dependent on its implementation.  Thus, signals do not so much "arrive" as simply change between high and low levels at particular times.

Thus, when one input goes high before the other, the gate is not yet activated, but there is not reason for it not to activate when the other goes high as well, as long as the first signal hasn't dropped to be low again in the mean time.
Now, with a biological system such as this one, it can also get messy because the high and low levels may not be cleanly separated and there may be significant leakage when only one is high.  In the case of the particular system that you are interested in, it looks like there's significant leakage for many of the configurations, so in many cases the second signal need not even arrive for the system to be at least partially on.


text:
How do you do Polymerase Chain Reaction (PCR) if you don't have a thermal cycler?
PCR has three steps: Denaturation, Annealing, and Primer extension. Let's say you do not have a thermal cycler in the lab, how would you mimic similar conditions and perform PCR?

three water baths and patience

Worth noting that three reasonably accurate water baths might cost more (and definitely take up more space) than a small, cheap thermal cycler

Use of 3 water baths was the original method. Perhaps isothermal amplification an option? Many reagents change colour upon amplification to visually identify successful pcr, which could make screening faster.


text:
Does ribonuclease processing of pre-crRNAs happen co-transcriptionally?
I understand CRISPR-mediated bacterial immunity to occur in the following simplified steps:

A CRISPR array is transcribed from promoters in the leader sequence to yield a precursor CRISPR RNA (pre-crRNA).
The pre-crRNA is processed by ribonucleases (e.g. Cas6 in Type III systems) to create mature crRNAs.1
crRNAs are loaded into Cas proteins to create an active CRISPR-Cas complex.

My question pertains to the timing of steps 1 and 2. Is there any evidence for co-transcriptional processing of pre-crRNAs? Put another way, must the full pre-crRNA be transcribed prior to its cleavage by ribonucleases, or can single crRNAs be cleaved from the 5' end of the nascent pre-crRNA. My understanding is that cas6 recognizes and cleaves at the base of the repeat stem-loop,2 and that these stem loops form spontaneously after a repeat is transcribed, so my hunch is that co-transcriptional pre-crRNA cleavage probably can occur, though I'd like some evidence from the literature to support/refute my intuition. I realize mechanisms of CRISPR immunity are quite diverse, so I'd appreciate references pertaining to any CRISPR system.
Some search phrases I've tried in Google:

co-transcriptional processing of crRNA
co-transcriptional cleavage of crRNA
crRNA processing concurrent with transcription
ribonuclease processing of nascent pre-crRNA
cas6 proximity to elongating rna polymerase

References

Carte J, Wang R, Li H, Terns RM, Terns MP. Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes. Genes Dev. 2008 Dec 15;22(24):3489-96.
Sokolowski RD, Graham S, White MF. Cas6 specificity and CRISPR RNA loading in a complex CRISPR-Cas system. Nucleic Acids Res. 2014 Jun;42(10):6532-41.

What difference will it make in the systems you are considering if there is co-transcriptional processing?

It may not make a difference. This question pertains to my research interests and has implications for the interpretation of my data.

I don't know the answer myself, but if it would make a difference in the interpretation of your data, it seems like that might be a good hook to build off of.

Thanks, @jakebeal. I'll take that into consideration if I don't get any answers in the next few days.

I originally posted this question in an attempt to understand a pattern I observed in my sequencing data. In the time since, I've formalized my interpretation as part of a manuscript, now posted on bioRxiv.
Some background: we performed precision run-on sequencing (PRO-seq) on bacterial cells derived from human stool samples. In short, PRO-seq involves supplementing permeabilized nuclei (or cell lysates, in the case of bacteria) with biotinylated NTPs, such that endogenous RNA polymerases incorporate biotin onto the 3' ends of nascent transcripts. Transcripts are then precipitated on streptavidin beads and sequenced, revealing (1) which genomic features are being actively transcribed and (2) the genome-wide and strand-specific positions of RNA polymerase with base-pair resolution.
Looking at the PRO-seq reads mapping to our metagenome-assembled genomes, we observe a wave-like pattern at loci annotated as CRISPR arrays. Figure 3 from our preprint gives one example:

(A) Coverage of PRO-seq and RNAseq reads across a CRISPR array in a US2 Prevotella sp. contig. Shaded boxes represent repeats. The large black arrow in each panel represents the leader sequence containing a putative promoter. Small black arrows in the “PRO-seq 5’ end” panel correspond to the predicted site of crRNA cleavage proximal at the base of the repeat stem loop.
(B) Predicted crRNA repeat secondary structure. The black arrow points to the phosphodiester bond that is likely cleaved by Cas5d during pre-crRNA processing, which marks the same position in the repeat as the small arrows in (A). The sequence logo shows perfect conservation of the repeat sequence for this array.
(C) PRO-seq captures nascent transcription of cas5d upstream of and contiguous with the CRISPR array.

Looking specifically at the position of the 5' ends of PRO-seq reads (bottom panel of A), we noticed a pile-up at the same position within each repeat corresponding to the putative crRNA cleavage site at the base of the repeat stem loop (arrow in B). Notably, transcription across this array coincides with transcription of a cas5d endoribonuclease gene upstream of the array (light blue rectangle in C).
Coming back to the title question...

Does ribonuclease processing of pre-crRNAs happen co-transcriptionally?

... I believe the answer is yes. Wave-like transcriptional profiles at CRISPR arrays are not a novel observation (see Figure 1 from Richter et al. 2012), but, importantly, our data include only nascent transcripts. This implies that crRNA cleavage by Cas5d is concurrent with pre-crRNA synthesis, at least in this human-associated Prevotella species.

Very nice answer as usual. Unfortunately the link for your manuscript doesn't seem to work (I'm being sent to a "DOI Not Found" page).

@tyersome thanks for catching that. Fixed


text:
Does vaccination lead to short-term secondary infection suceptibility?
For clarity, here is a summary of my question, per anongoodnurse's comment:
Does a lower peripheral lymphocyte count resulting from recent immunization render us more susceptible to infection by other viruses?

There are many bad-faith arguments circulating that suggest COVID-19 vaccines are doing more harm than good. One talking point is that COVID-19 vaccination leads to a temporary reduction in circulating white blood cells, leaving vaccinated individuals susceptible to infections in the short term.  Former NYT reporter Alex Berenson has been making such claims on Twitter:

1/ The fact the mRNA vaccines transiently suppress lymphocytes after vaccination has come up repeatedly on the feed as a possible explanation for post-vaccination deaths.
Fact check: This is TRUE. @BioNTech_Group
acknowledged it its papers (both 162b1 and 162b2 did so)

2/ By the way, they also lead to a sharp rise in C-reactive protein.
Would love immunologists or virologists to chime in on whether these changes may be in any way clinically meaningful

As sources, Berenson cites two papers detailing the results of phase I/II trials for mRNA vaccine candidates.1,2
Derek Thompson addresses this Tweet and other claims made by Berenson in an article in The Atlantic 3, in which Thompson asks experts to challenge the misinformation shared by Berenson. Regarding the Tweet above, virologist Shane Crotty said the following:

The claim he is making is simply fearmongering, connecting a simple physiological event with bogus claims of deaths ... The observation of lymphocyte numbers temporarily dropping in blood is actually a common phenomenon in immune responses ... The cells are not gone. They come back to the blood in a few days. It is generally a good sign of an immune response, not the opposite.

While Berenson's claims regarding post-vaccination deaths are derived from his (willful?) misinterpretation of the data, Crotty's response got me thinking about the immunological dynamics of vaccination. My question: Does the localization of lymphocytes to secondary lymphoid tissues lead to reduced circulating lymphocytes and a subsequent increase in susceptibility to secondary infections? By secondary infection, I mean a challenge from a pathogen that is different from the one for which the vaccine was received.
For example, a person receives a dose of one of the SARS-CoV-2 mRNA vaccines, then, sometime during the critical period when circulating lymphocytes localize to the site of vaccination, the same person comes into contact with an influenza strain for which they have no adaptive immunity.  Would that person have a lower chance of contracting flu had they not received the SARS-CoV-2 vaccine, all other factors being equal?
I found one publication that shows reduced risk for infection by influenza in patients who already had SARS-CoV-2, though this is attributed to pathogenic competition.4 Considering total pathogen dosage, it is logical to me that challenge with two pathogens is more likely to lead to illness than challenge with a single pathogen, but is there anything in the literature that suggests co-infection by an immunogenic "mock" pathogen (vaccine) and a secondary pathogen leads to a greater incidence of infection by the secondary pathogen?

References

Mulligan, M.J., Lyke, K.E., Kitchin, N. et al. Phase I/II study of COVID-19 RNA vaccine BNT162b1 in adults. Nature 586, 589–593 (2020).
Sahin, U., Muik, A., Derhovanessian, E. et al. COVID-19 vaccine BNT162b1 elicits human antibody and TH1 T cell responses. Nature 586, 594–599 (2020).
Derek Thompson. The Pandemic’s Wrongest Man. The Atlantic, 1 April 2021.
Stowe, J., Tessier, E., Zhao, H. et al. Interactions between SARS-CoV-2 and Influenza and the impact of coinfection on disease severity: A test negative design. medRxiv 2020.09.18.20189647.

Not an answer because I'm no immunologist at all (virologist though...) but the lymphocytes don't localize to the vaccination site, rather to secondary lymphocytic tissues - see here. The lymphopenia is a common occurrence in infection and vaccination and does not affect the antigen presenting cells already present from previous infections, only the pre-differentiation cells

Thanks for your insight, bob1! I'll correct my question. If you're willing to expand on this in an answer, there's a bounty up for grabs.

Unfortunately that's about as far as my knowledge of immunological processes goes. However, I think you will find that the answer is likely no - we are constantly under challenge from pathogens - think of all the bugs in your gut (E. coli) and on skin (Staph infections), inside your throat (strep) etc., and we don't see a sudden rise in these infections with vaccine programs. Note that anti-viral responses (CD8 T-cell) are slightly different to bacterial (CD4), though I don't know at which stage these play a role, though I suspect after the antigen-presenting stage.

@bob1 -  I would imagine that a significant challenge (what I imagine the OP is alluding to) would pertain only to response to another viral illness, not a bacterial infection, though the NYT reporter may not be implying that.

@acvill - Your question is a tiny bit misleading; yes, the number of lymphocytes circulating in peripheral blood decreases, we know that. The question should be (imo), "Does a lower peripheral lymphocyte count resulting from recent immunization render us more susceptible to infection by other viruses?" I can't answer that authoritatively, but decades of clinical experience leads me to lean towards "no". Interferons may be part of the reason. (I'm neither immunologist nor virologist, just a lowly FP/EM.) +1 for a well presented question.

Mechanisms exist that could explain a higher susceptibility to virus B after or during a an infection of virus A, e.g. via targeted inhibition of anti-viral cellular mechanisms. In general, however, these anti-viral mechanisms favor an anti-correlation between infections of different viruses; e.g. up-regulation of interferons drastically reduces the probability of viruses to successfully complete their replication cycles. I once discussed with a leading virologist the possibility to use interferons as a general preventative treatment to temporarily prevent the spread of viral pandemics; I was told that this would theoretically work, but cannot be done due to enormous side-effects associated with them, i.e. ill feeling and depressions.
Similarly, activation of immune cells and the complement system by interleukins and C-reactive protein (CRP), that you mentioned, generally promote resistance against subsequent infections. CRP also promotes chemotaxis to the site of infection, which is why I would, similar to your source, understand chemotaxis as a sign of a good immune response. Examples like this are listed in the paper:

Mold et al. (69) showed that CRP provides mice with protection against infection by the gram-positive pathogen Streptococcus pneumoniae by binding to a PCh determinant of the pathogen cell wall and activating the complement pathway.

The  complement system is also known to be effective in killing cells that are infected by viruses. Unfortunately, I could not find dedicated publication that connect lymphocyte migration with viral susceptibility, but my intuition is that lymphocyte migration should not be problematic when it comes to detecting viruses although it might contribute to the fact, that chronically elevated CRP levels are associated with the risk of cancer.
Importantly, one should not ignore behavioral aspects related to illness and vaccinations: There were reports of statistics, that people had significantly lower chances of being infected with CoV in the weeks between scheduling and receiving their first CoV shot, which was explained by individuals avoidance of risk. Reversely, people might loose their inhibitions concerning social distancing once they feel protected by vaccines, which could show up as a pseudo-susceptibility in statistics.


text:
Does cell phone radiation promote longevity?
This preprint presents results from a 2-year National Toxicology Program study on the effects of cell-phone radiation in rats. On page 8 of the PDF, survival results are presented. The presentation of the results is complicated by the fact that some treatment groups experienced significantly different effects when compared to the control group, while in other groups the effect was small but not significant. However, the gist is that rats treated with phone radiation lived longer, and that the effect is stronger in male rats.
I was confused by the fact that this result is presented in the "Results" section on page 8, while the seemingly less significant finding - that treated rats had higher rates of cancer or tumors - was presented in the "Summary" section on page 4. Obviously the study was funded to address concerns we have about observed relationships between cell-phone use and cancer, but when an effect on longevity was discovered, shouldn't this have become the primary result?
The plots on page 11 (Figure 3) and 12 (Figure 4) show some more interesting relationships between the survival of the various groups. Figure 4A shows the high-dose (6W/kg) CDMA male group overtaking the control males only towards the end of the study. With the GSM males (Figure 3A), as well as the low-dose CDMA males, the control group does worse almost from the beginning.
I'm curious to know why more people haven't commented on this result in news outlets. My opinion is that controlled experiments in animals aren't perfect, but they're much better than cheap "longitudinal" studies in humans. As far as I can tell, this is the best evidence we have to date, and it says that cell phone radiation promotes longevity. So why aren't we being required to sleep with phones next to our heads?

I don't think it is useful or relevant to discuss preprints. Wait until they have appeared in peer-reviewed journals.

@David There are actually a bunch of reviews at the end of that document. Most take (quite negative) issue with the study design and power.

If you read the discussion, and also the full contents of the PDF which include some reviewer comments, there is concern about the control group:

The survival of the control group of male rats in the current study (28%) was relatively low
  compared to other recent NTP studies in Hsd:Sprague Dawley® SD® (Harlan) rats (average 47%, range 24-72%).

Specifically, the control group had an abnormally high mortality rate for that strain of rat. There is no increase in longevity if you compare to historical data.

Yes, I read that. Is your argument that we should (a) do away with control groups and just compare with non-randomized controls from historical rat populations used in different studies in other parts of the world? Or that we should (b) use control groups but ignore the results when we disagree with them, falling back on historical statistics?

Because a randomized trial gives us a p-value, which, although not reported by the authors of this study, tells us that the in case of the null hypothesis, where the treatment is not doing anything, the observed differences between groups must indicate that a e.g. 1 in 20, or a 1 in 1000, event happened. And I thought the point of statistical analysis was not to fall back on explaining our results as due to extreme randomness.

@Metamorphic No, a control group should still definitely be used. p-values don't prove truth, they tell you when your results are more different than you would expect if there is no real effect, with some reasonable margin for error built in. You always have to use other sources of information at hand to interpret your results. It would already be a little extreme to change behavior based on just one study, and even more extreme to change behavior based on a study where there seems to be a significant problem with the control group.

@Metamorphic Not to mention the study also showed more tumors in the test groups than the control group, though that is also suspect because of the short lifespan of the controls. Truthfully, it seems like whether by randomness or some unknown factor not accounted for in the study, this work will have to be repeated with a new set of subjects to provide a result that can inspire more confidence.

I admit that the idea of changing behavior was a bit tongue-in-cheek - although for many people this would not be a change of behavior. But I'm not sure we're on the same page with the results - why do you say there is a "significant problem with the control group"? What would make it different from the other groups? They were all treated identically, except for radiation levels. Further, your quote indicates that the control group survival was not outside of the range of normal survival rates, so your use of the adjective "significant" seems misleading given the context.

Well, they gave a full range, not an interquartile range, and I am assuming they are basing that on many studies. It could be that only 1 study out of 1000 had lower survival than this one. It was sufficiently significant for both the authors and the reviewers to comment on it being an issue with the study. As far as the control group, we don't know precisely how the control group was treated. The researchers no doubt tried to treat the control group equally, but maybe they did not. Maybe control animal cages were in a row and disease spread between them when they were handled.

...the point is, we don't really know, but the control group is enough of an outlier from other studies that we should think more carefully about the results. You are much more likely to make a type 1 error if the control group itself is in the lower end of normal, and in fact this sort of thing happens all the time, especially if you consider a p-value of 0.05 to be significant. Part of your question was asking why this study isn't getting more play and why the authors didn't focus on this point: I think it's because they are not confident it is a real result, and they expressed that concern.

Thank you for thinking about this. I think that what it boils down to is, the authors don't give us enough data to make sense of their results. This is somewhat frustrating and makes me wonder what is being covered up - whether it is experimenter incompetence, as if the control group rats were all "in a row" as you say; or, alternatively, if it is a result which might threaten their National Toxicology Program funding, such as "X is not toxic".

@Metamorphic don't look for conspiracy where there likely is none. I personally applaud the researchers for publishing mixed results - that's not done often enough, and skews the literature (and our understanding) to a potentially large degree. Not every study will give perfect, absolutely clear-cut results. We don't know a lot of things, but that doesn't mean they're being covered up intentionally. Maybe it was a poorly-trained grad student. Maybe it was disease, or the animal facility staff, or the facility itself, or the supplier, or a million other possibilities. Don't be paranoid.

@MattDMo: "Don't look for a conspiracy"? "Don't be paranoid?" I don't appreciate your patronizing generalizations. Everyone has bias and you know very well that we can discuss that here. If you're so interested to distinguish between bias and incompetence, don't write to me. Write to the authors and ask for their p-values. Maybe you'll have better luck than I did.
I don't even know why you would bring up conspiracies in this case. The bigger "conspiracy" would be if they had bias in the other direction.

@Metamorphic I don't think MattDMo meant any harm. You are coming here and saying "Why aren't people taking this study more seriously?" and we are giving you a very good reason: because the data are suspect. That doesn't mean you throw out the results. I don't think the researchers are incompetent, as you suggest, I think they are pragmatic and reasonable: something in their results looks funny, so they aren't putting much stock in it until it gets confirmed. That's responsible research. I also wouldn't say the NTP's funding is dependent on finding particular things toxic.

@MattDMo makes a great point as well about the skew in the literature: that many studies go unpublished when the results are suspect or borderline. That's actually a big problem. It's much better to publish the results, with the appropriate caveats noted, so that future studies can take it into consideration. If you zap two more studies worth of rodents with cell phone radiation and their control groups suffer again, we can start to conclude that the radiation is making telekinetic rats that suck the life force out of their kin to prolong their lives. Until then, let's be cautious.

@Bryan: If you or Matt write asking for p-values and quantiles, that would be great. They exist, and at this point getting them should be easier than theorizing about our beliefs about other people's beliefs. ... Until then, I like your last hypothesis :)

@Metamorphic The paper hasn't even completed the peer-review process; the reviews included in the pdf you linked have already asked for some additional statistical presentation of the results, including p-values. I suspect all of these will be in the final publication.

@Bryan: I think that the pre-print phase is a great time to correspond with the authors of a paper. If you want to wait, that's fine. Just remember that the purpose of getting those numbers is primarily to validate the assumption in your answer, that certain deviations were significant. My question is based on significances already validated by the reviewers (e.g. p. 53). You could also replicate what the authors did by tracking down recent NTP SD-rat studies. Then collate the control group survival rates, and run a t-test. If you get say p < 0.05 then I would accept your answer.

@Metamorphic Did you read the entirety of the PDF you linked in your original question? NIH reviewers have already raised these issues, and uniformly show concern about the survival rates. Page 53 and similar was simply checking that the statistical calculations were performed correctly, and says nothing about the reviewers opinions of the data themselves, which they clearly feel are suspect.

@Bryan: I'm not interested in counting the number of reviewers that expressed concern, what's more interesting, for your argument, is counting the number of rats that survived and relating it to other studies. Is the relationship statistically significant?

Let us continue this discussion in chat.

"I'm curious to know why more people haven't commented on this result
  in news outlets"

Because for once the news outlets appear to be behaving responsibly, and waiting until the results are published in a peer-reviewed journal. You could do worse than follow their example.

@AliceD — I am sorry but it answers the part of the question I quoted. It is not a request for clarification nor a critique (in the correct or incorrect usage of the word), and it is in no sense suitable for a comment. In any case this is an old question that has only resurfaced because someone has dug it up. Not worth a flame war now.

I'm curious to know why more people haven't commented on this result
  in news outlets.

This is an easy point: nobody is interested in publishing this. A quick scan of my (not that bad) local newspaper shows stories in a few categories:

Photogenic stuff. About the moon, Antarctica, animals etc.
Male/female stuff, why we are not the same, or why women are better at something
Everything about food, medicine and vitamins
Climate change

Besides that, the story has to be news. It has to have some actuality, fit in with previous/further stories people are talking about, and people have to want to read it.
This story has none of that. Nobody is talking about cell phone radiation, and people are already convinced that it's not dangerous. 15 years ago this would definitely be on science pages everywhere.
Then there's also the science. Apart from what people are saying in the comments, you just cherry picked a single data point from a single study. The study was not designed and powered to show that cell phones increase survival, the authors don't focus on it (rightfully so), and there's also no plausible mechanism how this should work.
You can have a theoretical discussion about whether or not this fact is a reliable fact, and about whether or not the authors did everything right. However, I would put all my money on future research showing no enhanced survival, because circumstantial evidence is pointing in that direction and there's no mechanism.

Good first half, but... "you just cherry picked a single data point from a single study" - What are the other studies? I mean, other studies that would provide evidence about survival effects. "The study was not designed and powered to show that cell phones increase survival" - not designed in what way? "circumstantial evidence is pointing in that direction" - good if true, but can you elaborate? Thanks

It's highly likely there are other studies where animals are exposed to cell phone radiation. Probably there this effect is absent or not significant. Not designed / powered: if there is an effect it's likely that it's (extremely) small. By doing the study with low power, you're very likely to get either nothing (expected) or, if you get a statistically significant effect, the effect size will be overestimated. The circumstantial evidence is that similar mice in identical control groups that usually live longer.

See #3 in my question. If you think another study is relevant, I need you to track down the citation. Otherwise you're asking anyone who disagrees with you to cite every study ever published and show that it doesn't meet your advertised criteria. Some estimate that a total of 50 million journal articles have been published to date. It's much easier if you just tell us where to look.

You were interested enough to look this up in the first place, why would you stop looking when you find one number that fits what you want to think?

I don't appreciate your condescension. I made a simple request. Please back up your claims, or don't participate here.

@Metamorphic VonBeche is not condescending you. Every single person who has answered or commented here has roughly the same opinion. You are here asking for our help - no one is being paid here to do work for you. Sometimes it's helpful to teach by giving the answers, but more often its best to give someone the tools to do their own work on the subject.

I don't have time to play your games @Bryan. This page is for people who want to answer my question. If you think it would be a waste of your time to do that, or if you think that you would be hindering me from "getting tools", then either way you don't belong here. It's a Q&A site.

Your question wasn't about the dangers cell phone radiation, it was about why nobody was talking about this research and why the WHO doesn't advice us to sleep with our phones. The answer to that is just that it's not interesting and that the result is probably wrong. If you want to know more about this field of research I can recommend you to read some more literature and look up some reviews. To refer to the guidelines: "some thorough searching for the answer is expected". You came here with one cherry picked data point.

all_licenses: Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/


text:
Do any known viruses contain "junk" or parasitic genomic information?
After reading about so-called "mystery proteins" in this excellent summary of the coronavirus genome (and acknowledging that the "mystery" simply reflects our lack of knowledge about a very new virus), I'm curious if any known viruses can contain parasitic genetic information "along for the ride", or if their genomes are selected, generally speaking, to be as informationally efficient as possible, for what is encoded? In other words, does selection pressure allow for these parasites to contain their own parasites, and are there known examples of such, if so?

Here’s something: https://bmcbiol.biomedcentral.com/articles/10.1186/s12915-015-0145-1

That's a great answer, cheers for that.

I'm curious if any known viruses can contain parasitic genetic information "along for the ride"

Analogous to the parasitic transposons mentioned in the paper linked by canadianer, phage-inducible chromosomal islands are mobile elements that "hijack" the molecular machinery of bacteriophage during lytic infection of Gram-negative hosts.  PICI elements, as they're called, encode Rpp proteins that complex with the phage TerS homodimer, changing the conformation of the complex such that the PICI cos mimic is preferentially bound and packaged into the empty capsid.  Fillol-Salom et al. 2019 1 discuss in detail the mechanism of PICI propagation and provide a good graphical abstract of the process:

Similar elements exist in Gram-positive bacteria, specifically Staphylococcus aureus, called Staphylococcus aureus pathogenicity islands (SaPIs).  Instead of encoding a separate protein that complexes with the phage TerS, SaPIs encode a homolog of TerS that specifically recognizes and packages SaPI DNA into phage capsids.2
Note that this answer does not directly address the question of "junk" DNA in viruses, as PICI elements are dormant in the genomes of the bacterial hosts rather than the genomes of the viruses themselves.  Though, I believe this is relevant insomuch that they parasitize bacteriophage, and are therefore "along for the ride" in the viral capsid.

Citations

Hijacking the Hijackers: Escherichia coli Pathogenicity Islands Redirect Helper Phage Packaging for Their Own Benefit
Role of Staphylococcal Phage and SaPI Integrase in Intra- and Interspecies SaPI Transfer


text:
What is a "monomeric polypeptide"?
In the sentence: "Bacteriophage (viral) polymerases are typically monomeric polypeptides".
I know that polypeptides are chains of amino acids monomers. But what is a "monomeric polypeptide" and what other kinds of polypeptides are there?

You're right to highlight this confusing statement. As David's answer points out, this is a case of tautology. The term "monomer" or "single polypeptide chain" would be more appropriate I believe. It's a very minor point though and the sentence does technically make sense.

Monomer in this context means the protein has only one polypeptide chain. In some  proteins, after the individual chains have folded into their 3D or tertiary structure, they associate (generally non-covalently) into a higher order or quaternary structure. A protein with a quaternary structure containing  two copies of the same chain would be called a homo-dimer. One with one copy of each of two different chains is a hetero-dimer.
Haemoglobin is a well-known example of a protein with quaternary structure — it has two copies of the alpha- and two of the beta- chain. Further elementary treatment can be found in Berg et al. and explanation of the nomenclature in this Wikipedia page.
Monomeric polypeptide is somewhat misleading in the sentence quoted. Monomeric protein would have probably been better as you wouldn't be likely to say ‘dimeric polypeptide’, given that polypeptide implies a single chain.

A small doubt: What would you call mature insulin? It has two polypeptide chains covalently linked via disulphide bonds. It is a monomeric protein, however.

@WYSIWYG I wouldn't use my first sentence as a definition of monomer, just as an explanation of what the writer meant. In talking about insulin I personally would say that although it has two polypeptide chains it consists of a single globular subunit and therefore cannot be said to have quaternary structure (as I would define it). This is easy to justify since we now know that the general tertiary structure of insulin is formed by the folding of a single polypeptide chain, proinsulin, which is cleaved to activate it (with a small but significant change in tertiary structure).


text:
Why are minor introns bounded by nucleotides AT-AC, where T is not normally in RNA?
Major intron boundary nucleotides are GU-AG. Minor intron boundary nucleotides are AT-AC. Why does the former use RNA Uracil and the latter use DNA thymine? Since introns are in the primary RNA transcript, shouldn't both be RNA and use U rather than T?

It is not unusual for RNA sequences to be reported with T instead of U; the writer is reporting the cDNA sequence instead of the RNA sequence.  In RNA at the boundary of the minor intron, there is a uracil.  In the following page, the writers follow the oft-used convention of describing RNA with T instead of U (writing the cDNA sequence).  https://en.wikipedia.org/wiki/Minor_spliceosome


text:
DNA is charged negative. Where is all the positive charge in my body?
DNA is charged negative because of its phosphate backbone. Since charges need to be balanced (so that there are no charges building up somewhere), what is the positive charge which neutralizes this negative charge?

It would be great if you state the context of the question to allow a more constructive response. Many ions such as Na+ and Mg2+ are positively charged.

There is no noteworthy context. I'm a physicist, just saw the structure formula of DNA and was wondering about this.

@Bez The question is short and it is ok to ask it out of curiosity.

Another point to be noted is that isoelectric pH of DNA is close to 7 (6.7) [Ref].

DNA in the body is not available as a free molecule, it is organized around DNA binding proteins, mostly the histone octamers. These proteins carry positive charges (mostly from lysine side chains) which interact with the phosphate backbone of the DNA. It is like in the figure below, the charges play an important role in the tight packaging. See here for more details.

The charge (or the temporary neutralization at some points of the DNA) is also important for the regulation of the DNA. Acetylation neutralizes the histone tail and loosens the DNA-protein structure (see here for more information).
Positively charged ions don't play a role in neutralizing the DNA charge in living cells (this is different for precipitations), as both are solubilized and split into ions. The concentration of positively charged ions like sodium and potassium are tightly controlled by the cells, as they are important for the function of transporters (for example the sodium-glucose transporters and others) or for keeping up membrane potentials. Moving the ions, which have to neutralize the DNA charge around would lead to a polarized nucleus.

Your cells have a high concentration of sodium ions that are positive. Other important cations are potassium and calcium. Additionally, many amino acids are positively charged at physiological pH. DNA is not the only source of negative charge in your body, other amino acids are negative, and most cell surfaces are negative as well. The interactions between charged species play an important role in many biochemical processes, but at the end of the day, and for the whole organism, the charges are all pretty much balanced out.

Could you please provide a reference regarding the statement "the charges are all pretty much balanced out"? I could be wrong but the resting membrane potential for example in neurone is -70mV so that doesn't sound like balanced!

That's charge of inside vs outside of cell. If your whole body had a significant net charge, you'd be constantly affected by static cling, or sparking static electricity all the time, or electrostatically repelled from other living things. Based on my observations, these things don't happen.

I agree! but at a single cell level cells can have a charge so perhaps it would be best if you edited your response slightly to clarify the last sentence.

Made an edit to clarify the statement is about the whole animal.

The negative charge in DNA is localized to individual oxygen atoms in DNA. This negative charge is balanced by positive ions in the body.
In the case of a magnet or molecules with a dipole, negative and positive magnetic field lines align to produce an overall magnetic field. DNA has many small dipole moments but none across the entire molecule.
In the case of a neuron, an excess of negative charge inside the cell and positive charge outside the cell is maintained by the diffusion gradient of potassium and sodium across a membrane. An overall electric potential is not created with DNA.


text:
Number of homozygous recessive offspring
A question from the KVPY-SX examination, organised by the Indian Institute of Science (IISc) in Bengaluru, India, held on 3rd November, 2019.

A plant heterozygous for height and flower colour (TtRr) are selfed
and 1600 of the resulting seeds are planted. If the distance between
the loci controlling height and flower colour is 1 centimorgan, then
how many offspring are expected to be short with white flower (ttrr)?
(A) 1 (B) 10 (C) 100 (D) 400

The answer given by the organising committee, of the KVPY, is (A).
My problem with this answer is that if the alleles are in a cis arrangement*, and the distance between them is 1 cM, then the recombination frequency will be 1% (a good enough approximation in this case, in my opinion).
So, on selfing of TtRr, we'll get;
TR - 49.5%
Tr - 0.5%
tR - 0.5%
tr - 49.5%
So, the probability of getting a homozygous recessive genotype, which we obtain from tr × tr, is (0.495)^2 = 0.245, and hence, we get (0.245)(1600) = 392 offspring approximately, the answer closest to which, is (D).
*By "cis arrangement" of alleles, I'm assuming that the 'T' & 'R' alleles are present on one chromosome, and 't' and 'r' alleles are present on another. (An NCBI webpage explaining this terminology)
Where am I going wrong?
What's the right solution to this problem?

Why do you assume the alleles are in a "cis arrangement"? We typically talk about alleles being linked, but you need to specify which alleles are linked (e.g.s 'T is linked to R' or 'T is linked to r'). Is the former what you mean by "cis arrangement"? Also, have you included the full text of the question? It is quite possible that this is (yet another) example of a poorly worded question ... ——— Also note that this is a "homework" question (see [help] for details about what that means. You've shown the required effort, but you must also include the homework tag on these questions.

@tyersome Thanks for the suggestion; I have included the homework tag now. What I mean by "cis arrangement" is that the 'T' and 'R' alleles are present on one chromosome, and 't' and 'r' on the other. Yes, I've included the full text of the question.

You're welcome. Can you please also edit in answers to the questions I've asked? With the information you've given I'm not sure that any of those answers is correct ...

@tyersome you can consider posting an answer justifying why you are not sure of any of those answers being correct, and  the appropriate approach towards this, given the right (amount of) information.

I don't see anything in the problem statement that allows assuming cis or trans arrangement.

@mgkrebbs What arrangement other than these two can we assume? All I'm saying is that, if the alleles were in a cis arrangement, their answer doesn't hold true. This in itself is enough to invalidate it.

@ShishirMaharana, "enough to invalidate it." -- invalidate the answer? No, the trans calculation results in an expected number between 0 and 1, so the "1" answer would be the best answer in that case.  Invalidate the question? Well, as shown, the question presentation doesn't have enough information to select an answer, so that's crappy.

I wonder if the creators wrongly modified a simpler question and botched the modification? If height and color are unlinked, the number of ttrr offspring should be 100/1600. Perhaps they wrongly decided "we'll make the loci 1cM apart, so the new result will just be the original result x 1%, or 1."

Taking your assumption of a cis arrangement of alleles to be true, here is how I would solve the problem.
A diploid plant heterozygous for height and flower color is selfed.
$TR / tr \times TR / tr$
A map distance of 1 cM means that 1% of meioses will result in recombinant gametes, so we can calculate the expected distribution of gamete genotypes assuming equal probability of the strictly dominant or strictly recessive gametes in the absence of recombination.
$p(TR) = 0.5(1 - 0.01) = 0.495$
$p(tr) = 0.5(1 - 0.01) = 0.495$
$p(Tr) = 0.5(0.01) = 0.005$
$p(tR) = 0.5(0.01) = 0.005$
The expected proportion of homozygous recessive $tr/tr$ offspring is given by the product of the gamete probabilities.
$p(tr) \times p(tr) = 0.495^2 \approx 0.245$
$0.245 \times 1600 = 392\space\texttt{offspring}$
So it seems that your answer is correct and the given answer is wrong.

Since the loci of interest are very close together, a quick way to think about this is to treat the height and flower color loci as a single locus with dominant and recessive alleles $A$ and $a$ (i.e. independent assortment never occurs). This allows us to model the problem as a monohybrid cross of heterozygous plants.
$Aa \times Aa$
The phenotypic ratio of dominant to recessive progeny from a monohybrid cross is 3:1, so you can expect 25% of offspring to be recessive.
$0.25 \times 1600 = 400\space\texttt{offspring}$


text:
Who invented dN/dS?
I am writing a paper, and I want to refer to the original paper that coined the term dN/dS (or Ka/Ks for that matter). I have found early works on dN and dS (like Miyata and Yasunaga 1980), but cannot find the first paper that used the ratio dN/dS. Does anyone know which paper is the best to cite dN/dS?
Edit: dN is the rate of non-synonymous substitutions per non-synonymous sites; dS is the rate of synonymous substitutions per synonymous sites. dN/dS is simply the ratio between dN and dS.
Reference: Miyata, T., & Yasunaga, T. (1980). Molecular evolution of mRNA: A method for estimating evolutionary rates of synonymous and amino acid substitutions from homologous nucleotide sequences and its application. Journal of Molecular Evolution, 16(1), 23–36. https://doi.org/10.1007/BF01732067

I would suggest that you write the full names of the variables (dN, dS) it might help to better understand the context of the question and get more answers

In the paper you cite, we see KA and KS defined by the authors.

The direct comparison between a pair of known
  nucleotide sequences in coding regions permits us to evaluate both the nucleotide differences
  per site caused by nucleotide substitutions leading to amino acid changes,
  KA, and those leading to synonymous changes, KS, simultaneously.

The same year, Motoo Kimura published A Simple Method for Estimating Evolutionary Rates of Base Substitutions Through Comparative Studies of Nucleotide Sequences, in which he derives similar terms k'(nuc)S and k(nuc)A.

Writing k'(nuc)S rather than kS' to emphasize
  that this refers to the rate per nucleotide site, we get, using Eq. 14, the following
  formula.
$k'_{(nuc)S} = -\frac{1}{4T} log_e (1 - 2P - Q)$
The corresponding formula for amino acid-altering substitutions may be obtained by
$k_{(nuc)A} = \frac{K"}{2T}$

The reason why neither paper cites the other is because they appeared in the same edition of Journal of Molecular Evolution.  Based on the extensive cross-citation between these authors, as well as some co-authorships, it's likely that the development of the KA and KS metrics was simultaneous, if not collaborative.


text:
Why can SPRITE measure many simultaneous interactions?
Why is the SPRITE method not limited to a number of simultaneous interactions that can be measured, while microscopy and proximity ligation are limited to 2-3 interactions?

You list three methods for determining the pairwise interactions of DNA loci.

SPRITE is a split-pool sequencing technique. First, samples are crosslinked to preserve DNA-protein-DNA interactions. Then, DNA is enzymatically digested to create free ends for barcoded adapter ligation. Crosslinked globules are "split" into many wells for barcode ligation, then "pooled" together again in a single tube. After iterative rounds of "splitting" and "pooling", DNA molecules are sequenced. Pieces that are covalently associated by crosslinking will have the same set of barcode sequences, implying that those loci are spatially associated in situ.
Like SPRITE, proximity ligation techniques start with chemical crosslinking and enzymatic cutting to create free DNA ends. In "all-against-all" proximity ligation (Hi-C), free DNA ends are joined together in a dilute ligation reaction. Paired-end sequencing of DNA molecules containing ligation junctions reveals interactions between distal loci. Taking into account distance-dependent interaction decay, the contact frequency of locus pairs can be used to identify potentially meaningful DNA-DNA interactions.
Microscopy-based methods are limited by spatial resolution and the number of fluorophores (colors) available on a given platform. However, clever multiplexing may be able to overcome the fluorophore problem.

In short-read proximity ligation sequencing, it is true that a single DNA molecule can only ligate to one other DNA molecule, whereas SPRITE allows association of a single DNA molecule with one or more other DNA molecules – however many were crosslinked in the same globule. In practice, however, Hi-C is performed on bulk cells, and therefore the same locus may associate with multiple different loci across the contexts of different cells. Moreover, long-read sequencing can capture multiple ligation junctions in a single read, allowing for the inference of primary, secondary, tertiary, etc. contacts.


text:
How is incomplete dominance explained at the molecular level?
What happens at the molecular level between two alleles that demonstrate incomplete dominance? Do the proteins coded by each allele have different functions, or is there an interaction at the RNA level?

Allele could mean different things: it can be a point mutation, a whole new gene or even a whole chromosome. The molecular mechanisms involved obviously depend on which of those we are talking about.

@RogerVadim My understanding of "allele" would exclude "a whole new gene or even a whole chromosome". Did you have some examples in mind?

@Armand From the point of view of population genetics allele is a rather general term, just like mutation is not necessarily an SNP. Btw, I didn't mean new chromosome - but that chromosomes inherited from the two parents may have many differences between them. Here is from Gellespie: "Here we will use locus to refer to the place on a chromosome where
an allele resides. An allele is just the bit of DNA at that place."

@RogerVadim OK, I guess we'll agree to disagree.

@Armand In order to meaningfully disagree you need to suggest your definition of an allele :)

@RogerVadim Good point. It would be something like "homologous DNA at the corresponding locus on the homologous chromosome" .  I agree that the origin of "allele" in pre-molecular genetics makes a molecular definition somewhat problematic.

@Armand Homologous is a good point - I guess my phrasing about new gene was misleading - at least for diploid organisms. My main point is that allele could be a rather big piece of DNA (or even several pieces) with complex functioning on molecular level. I would change nothing from the point of view of pop. gen., in particular, in terms of discussing dominance, but it may correspond to many different molecular mechanisms.

@RogerVadim Re: "allele could be a rather big piece of DNA (or even several pieces) with complex functioning on molecular level" It seems to me that an allele (being a gene) should be a single functional unit and should also exist at a single locus (i.e. be essentially in complete linkage disequilibrium with other alleles of the same gene). Once we start considering more complex molecular configurations, I don't think the term "allele" applies anymore. Is there a population genetics example with multiple pieces of DNA that you could describe?

@Armand recombination does not have to occur necessarily at the boundaries of genes. If a complex phenotipic trait is determined by a joint action of several genes, and these are separated due to recombination, this in itself could be mechanism for the incomplete dominance in question.

Molecular mechanisms of incomplete dominance (also called partial- or semi-dominance) vary.  As an example, let's look at snapdragons and morning glories, two flowering plants that both exhibit incomplete dominance relative to flower pigmentation.
Snapdragons (Antirrhinum majus) have multiple alleles at the nivea locus.  The wild-type nivea transcript (niv+) encodes a chalcone synthase involved in pigment biosynthesis, and a single copy of the niv+ allele is sufficient for wild-type pigmentation.  However, niv+ is semi-dominant with the mutant niv-525 allele, leading to reduced pigmentation in the heterozygote.  The original publication by Coen and Carpenter 1 describing this effect offers some possible molecular explanations:

Transcription factor sequestration.  The niv-525 allele could encode a defective enzyme.  This allele also contains an inverted duplication of a transcription factor binding domain required for enzyme expression.  The defective niv-525 allele therefore titrates away transcription factor from niv+, leading to less enzyme and therefore less pigment.

Transvection.  There is some physical interaction between alleles on homologous chromosomes, such that transcription of one or both alleles is activated or repressed in trans.  This could be realized as binding of the enhancer associated with the wild-type allele to the promoter of the defective allele.  Figure 9 of Gohl et al. 2 has a good explanatory graphic.

Anti-sense hybridization.  The inverted duplication on the niv-525 allele contains a TATA box.  Transcription initiated at this site would result in a transcript anti-sense to the wild-type mRNA.  Hybridization of the sense and anti-sense
RNAs creates dsRNA that is selectively degraded and/or inefficiently transported out of the nucleus, ultimately leading to less enzyme product.

Morning glories (Ipomoea purpurea) show similar genetics at the A locus, which also encodes a chalcone synthase.  However, unlike the snapdragon niv+ allele, a single copy of the wild-type a allele is not sufficient to produce wild-type pigmentation.  Therefore, incomplete dominance in morning glory results from a simple dosage-dependent mechanism, as described in Johzuka-Hisatomi et al. 3

References

Coen ES, Carpenter R. A semi-dominant allele, niv-525, acts in trans to inhibit expression of its wild-type homologue in Antirrhinum majus. EMBO J. 1988;7(4):877-883.
Gohl D, Müller M, Pirrotta V, Affolter M, Schedl P. Enhancer blocking and transvection at the Drosophila apterous locus. Genetics. 2008;178(1):127-143.
Johzuka-Hisatomi Y, Noguchi H, Iida S. The molecular basis of incomplete dominance at the A locus of CHS-D in the common morning glory, Ipomoea purpurea. J Plant Res. 2011;124(2):299-304.

Strictly speaking, incomplete dominance is an interaction between two alleles of the same gene not between two genes.
The most common cause of this is the dosage effect. For example, the product of CHS-D gene is an enzyme required for the synthesis of purple pigment anthocyanin in morning glory flowers. If a plant has two functional (A) alleles of this gene it produces enough pigment to have intensely purple flowers. The a allele has the loss-of-function mutation (isn't able to produce working enzyme) so homozygous a/a plants will lack purple pigment resulting in white flowers. But heterozygous A/a plants have half of the normal enzyme level and half of the pigment so the flowers are lighter purple (source).  Genes like this are said to be haploinsufficient. In cases of full dominance, one functional copy of the gene is enough to produce a "normal" phenotype (the gene is haplosufficient) or the gene is upregulated to bring the concentration of functional products to the required level (source).
There are other mechanisms for incomplete dominance. For example, in snapdragon flowers, the allele niv-571 at nivea locus can inhibit the expression of "normal" Niv+ allele in heterozygous plants (source).

Zlira, it seems you beat me to this answer by 30 seconds, with the same examples!

Could be missing something here, but incomplete dominance is more like an absence of interaction in both biological and statistical senses- e.g. the additive model is a case of incomplete dominance in which you model phenotypes by summing the effects of each allele independently. Dominance/recessivity is conversely best understood as an interaction (absence of independence) between alleles. As you say, dosage is a good mechanism for codominance, but dosage effects will be strongest in the absence of allelic interactions.

Dominance or Recessiveness is the characteristic of allele, not gene.
Particular gene contains data for producing specific protein.  Protein produced from one allele can effect protein synthesis of other one.   Protein formed from recessive allele can show dominance over the dominant allele, and this is known as Dominant negative.
The protein produced from one allele is faulty and prevents other allele's protein to work properly. Both the proteins interact with each other in particular cell.
Genetic trait for red hair is an example of dominant negative.   Gene MC1R is responsible for red hair. Protein produced by this gene converts red pigment into brown.  When this protein is faulty the red pigment is accumulated and hair appears red in colour. Usually red hair is recessive trait.  Two faulty MC1R gene results in read hair characteristic.  Both the proteins formed from MC1R gene need to bind so that red pigment converts into brown.  But when functioning product binds with faulty one, the protein becomes defective.  So this defective MC1R protein is responsible for showing dominance.
https://genetics.thetech.org/ask-a-geneticist/genotype-vs-phenotype

Thanks....The answer is not related to incomplete dominance.

It is actually answer to second half of the question..as incomplete dominance is explained in earlier answers.


text:
Is my understanding of the Bergmann's rule correct? (mass is secondary)
According to Wikipedia, Bergmann's rule applies because animals living in colder areas have greater  surface-area-to-volume ratio. If I understand that correctly, the advantage of the bulkier bodies of the polar animals isn't really about having a greater mass but about having a shape more closer to a sphere - which usually means having a greater mass.
Am I right?

No, your argument seems to be flawed. Sphere’s surface-to-volume ratio is proportional to the radius, and a small sphere may have worse ratio than a bulky irregular body.

@jmster That's exactly what I don't seem to get. Isn't size relative? Like how come the ratio is different for a small ball and a big one. The way I see it, the size only tells us comparison to human metrics like meters. Let's say there's a planet A with a ball 1 meter wide and there's a planet B which is twice as big so the humans who live there say the ball is 0,5 meter wide even though it's the exact same size as ball A. Can you please explain how come "size" matters as a seemingly arbitrary parameter?

Heat transfer and many other physical processes depend on the molecular structure of the materials. The molecules on a big planet are not proportionally bigger than on a small planet, that's why you cannot expect everything that happens on those planets to scale up linearly.

@jmster Right, thank you! Both of the occasions when I was explained the rule involved the presenter mentioning that surface of a sphere grows with square of the ratio which rightfully didn't make sense to me as the explanation.

A couple of things. First, SA:V is lower in colder environments (see the similar "ecogeographic rule," Allen's Rule, Wikipedia. Second, SA:V and mass are two somewhat independent traits are the result of somewhat independent evolutionary mechanisms. Temperature affects mass, temperature affects SA:V, but SA:V does not affect mass.

@ChrisMoore Thanks, If I get you correctly, you confirm jmster's comment that the Bergmann's rule applies because it takes more molecules to build several small rabbits than to build one rabbit of the same mass?

@Probably : Not exactly. I am meaning to state what I have in the last sentence: temperature affects mass and temperature affects SA:V, but SA:V does not affect mass or vice versa.


text:
epitope prediction/ mapping
B-CELL EPITOPE PREDICTION
Regarding this article:

"Such a molecule can be synthesized or, in case of a protein, its
gene can be cloned into an expression vector."----- is a particular
line in this article. What does expression vector mean here??
"antipeptide antibodies"---- what are these?
"immunogenic peptides"----these are basically epitopes, am I right??

An expression vector is a (usually circular) piece of DNA which is able to replicate inside a (bacterial) cell independently of the genome. Vectors can be transferred between bacteria and can express proteins from their own DNA sequence. They are used to express proteins inside of bacterial cells which can subsequently be purified. In this case the sequence for an immunogenic peptide is cloned into a vector and expressed in bacteria.
These are antibodies which are directed against a certain peptide. This is one technique of generating these antibodies. Making a (synthetic) peptide and then immunize animals against it. Then you can purify the antibodies from the serum of the animal.
Yes. You make short peptides of 8-12 amino acids (which is the length that the antibodies binds to) and use them for immunization.

"independently of the genome" -did not understand this term.
"Vectors can be transferred between bacteria and can express proteins from their own DNA sequence"-This line means that one bacteria can carry the gene of another bacteria and hence express the proteins of its main parent bacteria in the new parent bacteria??
"ïmmunize animals against it." - meaning the newly created antibodies are injected into the body of victim so that they can fight that particular peptide??

Can you please ask this in a new question? It is easier to answer (since pictures can be added) and it is also considered more appropriate.

I could but if i ask, it will be closed down... so whats the point?

It is basically a piece of DNA which is independent from the cells genomic DNA. Have a look at the Wikipedia page, which also contains an image.


text:
What is the name of this plant? (found in a rock)
I found it in Western Turkey.It was growing on a rock so what's it?

I have been looking into this for days, but this plant is difficult to identify without its flower. I reached out to a botanist at Dartmouth, who suggests that it is either one of two species-- a nasturtium (Tropaeoleum sp.) or a geranium (pelargonium). The leaves are what are called peltate, meaning shield-like with the stem attached directly underneath.

Source:

Botanist at Dartmouth
http://etc.usf.edu/clipart/48400/48426/48426_peltate_leaf.htm

On further research, specifically of peltate leaves, this plant looks more like Umbilicus rupestris, which is commonly found in southern Europe and grows in damp rock crevices!
Source:

http://en.wikipedia.org/wiki/Umbilicus_rupestris

+1 - Great job! Key here is that the plant is a succulent (neither nasturtiums nor geraniums are). The succulent nature is easier to see in the OP's picture, where the leaves are slightly wilted.


text:
What are host cellular factors?
With respect to this paper:
Global Analysis of Host-Pathogen
Interactions that Regulate
Early-Stage HIV-1 Replication
What does the term "host cellular factors" mean??

Generally spoken, "host cellular factors" are proteins, receptors etc. of the host cell. In this case, they are important for the HIV infection. The authors write:

Recently, a genome-wide siRNA analysis revealed over 250 host cellular
  factors that influence HIV-1 infection.

This means gene silencing techniques where used to identify the host cellular factors which are important for the infection, you can find these results here: "Identification of host proteins required for HIV infection through a functional genomic screen."
The overall goal is to identify proteins which are important for the function and infection of HIV to understand the interaction between virus and host cell better.


text:
Klenow amplification
Am I correct in thinking that in a standard probe synthesis reaction where primer/template are heated up and then cooled down for Klenow to extend, the theoretical maximum amplification is doubling of the template?
Given that there is essentially only one melting/primer hybridization step, there can't be further amplification after doubling?
I understand that there could be edge cases. IE. random hexamers are used where initial amplification product is displaced by a further upstream hexamer. But given an idea situation where we have just one specific primer and one specific dsDNA template that primers can bind to, the maximum yield will be double?

Which version of Klenow — i.e. are you asking about the fragment with or without 3'→5' exonuclease activity?

Klenow seems to lack exonuclease activity ("proof-reading"), but otherwise works like normal DNA polymerase, so I wouldn't expect anything different from PCR. On each step, as long as there is enough template and primers, you'll get doubling of current template.


text:
Runs of homozygosity - degree of inbreeding and disease associations
Been reading recently about methods to determine how inbred a particular person is by measuring their "runs of homozygosity." Loosly, these are defined as regions of the genome (typically >1Mb) where the person is homozygous for all polymorphisms, suggesting their parents were identical for this region. If a person has 'many' of these regions, it suggests their parents were closely related.
A study in 2015 [1] found some evidence that people with more regions of homozygosity had higher risk of heart disease.
My question firstly relates to the amount of homozygosity you might expect in a 'normal' out-bred person, compared to an in-bred population (e.g. small community), and then to 'extreme' examples of relatedness (parent/child offspring). In my searching I didn't see any cut-offs, if they exist, or whether this is even the right way to be thinking about it. And secondly, just how bad is it to have these runs?

Christofidou P, et al. Runs of Homozygosity: Association with Coronary Artery Disease and Gene Expression in Monocytes and Macrophages. Am J Hum Genet. 2015; 97:228–237. PMID: 26166477.

FIRST QUESTION: It all depends on the allele frequency in the population you consider. Under the Hardy-Weinberg assumption, the probability of two randomly picked allele to be identical by descent is $\sum_i x_i^2$, where $x_i$ is the frequency of the $i^{th}$ allele. If you look at a region that is under strong purifying selection in a large population, then most SNPs will have very low minor allele frequency and very low heterozygosity and therefore you would see very long runs of homozygosity.

SECOND QUESTION: The article you cite is probably one of the best place to go to ask "how bad are those runs". They probably report an effect size at some point.