2,340 Matching Annotations
  1. Jul 2021
  2. Jun 2021
    1. Escherichia coli was not reliably distinguished from Shigella and other Escherichia species sharing the high 16S rRNA gene sequence similarity to each other

      Is this due to sequencing error rate of nanopore or the 16s are really that identical?

    1. Goals of this paper

      1. What are the barriers of HGT across organisms of increasing phylogenetic distance?
      2. What determines the host range of conjugative elements (ICE, MGE, plasmids etc.)

      Questions

      1. What does two plasmids being "related" mean to you guys? What does the sequence similarity or proteome similarity tell?

      Evidence for functional relevance of PTU

      1. PTUs have some correspondence with Inc groups and Mob types (both being more functionally informative) `
    2. ANIL20 refers to the total length of the shortest plasmid in the comparison.

      What does this mean if the shortest plasmid is really short - < 5 kb like the engineered plasmids and the larger plasmid is a real 50 kb plasmid? What does similarity of such a small region to such a large plasmid mean?

    3. This causes that otherwise unrelated plasmids show certain fragments of their genomes with high ANI values

      If they share the same MGE sequences, what else do you mean by otherwise unrelated? What does two plasmids being related mean to you guys?

    4. this trend dissipated when we looked within the Enterobacteriaceae family (Fig. 1e, f), indicating that the plasmid-encoded genome is widely shared among different genera in this family

      This could be an artifact due to proteobacteria being by far, the most studied family among bacteria?

    5. it is unclear whether, at the distal end of this phylogeny, there is anything similar to a “molecular species”: a group of genetically coherent genomes that evolve together.

      Considering that the concept of microbial species with functional similarity based on genetic similarity is itself debated. For example: There could be two strains, differing by a single nucleotide of some important gene, loosing a function with a huge consequence.. The same could be true for plasmids too if there are mutations in a particular origin of replication etc.

    6. determining the host range of a certain plasmid requires ways to establish which plasmids can be considered equivalent

      Or to determine the functional elements that determine host range and establish equivalence only within those sequences

    1. To separate cells from electronic background noise and abiotic particles, it is recommended to include nucleic acid stains

      Is a stain expected to be more homogenous signal compared to a fluorescent protein expression population of cells?

    1. Because of the technical complexity of manufacturing coronavirus vaccines, waiving intellectual-property rights, by itself, would have little effect

      Is this only relevant to the newer mRNA technology?

    1. However, even in unexposed barley seedlings 16% of the cells were induced after 24 h, suggesting the presence of other aromatic compounds were inducing xis-int expression from the regulatable tbuT promoter.

      or leaky expression of the Xis-int?

    1. two isogenic mutants of the bacterium Pseudomonas stutzeri.

      If they each have a deletion in individual genes, how can they be called isogenic?

      Their growth rates might also be different

    2. . Thus, we conclude that genetic variants of the consumer are unlikely to cause the emergence of the two patterns of spatial self-organization.

      The conclusion should be that this assay cannot test for the genetic variant theory unless variants can be isolataed prima-facie and the experiment reproduced with variant (of one or both strains) vs the original mixed population

    3. If the “consumer first” pattern were caused by genetic variants, then the number of “consumer first” patterns that emerge per range expansion should depend on the initial cell densities of the producer or consumer.

      The pattern is caused by the interaction of the two strains, so altering their ratio will also change the pattern even if it is not caused by genetic variants. How do you control for that?

    1. From this result, we considered that las promoter (BBa_J64010) was not regulated by lasR and 3O-C12-HSL.

      Could the RBS be too weak that expression is not seen?

    1. As the speed of unperturbed electrophoretic polynucleotide passage precludes the resolution of individual nucleotides, a DNA- or RNA-binding motor enzyme is added to the system to gain a processive and slowed down passage (millisecond scale per nucleotide) of the polynucleotide through the nanopore
    1. prepared for nanopore sequencing. This procedure involves end preparation, adapter ligation and incubation with the motor protein

      What is this motor protein, helicase? It is not mentioned in the nanopore ligation sequencing kit explanation. Does it come attached to the adapters?

  3. May 2021

    Tags

    Annotators

    1. simultaneously regulating transcription and translation, we show how basal expression of an inducible system can be reduced, with little impact on the maximum expression rate. Using this approach, we create several stringent expression systems displaying >1000-fold change in their output after induction

      From fig 4a: basal expression did not really reduce in 2 out of 3 stringent expression systems designed and seems to be already low in the native Ptac (black line). This is a misleading claim in the abstract which could have been worded better. file


      I do see that table 1 reflects basal expression as a percentage of the maximum expression, so maybe this is what is being referred to in the abstract?

    2. Both L2 and the gene of interest (GOI) are separately transcribed by PL1 promoters and the product of L2 activates translation of the GOI transcript.

      Repeating the same promoter Pl1 twice could cause evolutionary instability due to recombination. How to avoid that?

    1. mf-Lon does not recognize or degrade ec-ssrA, providing a protease and cognate degradation tag with orthogonal functionality in E. coli

      I wonder if this insulation will be applicable to other gram negatives? (or other gram positives too?)

    1. the very idea that you could "know" what certain mutations would do — i.e., that you'd know what you'd create and what effect it would have on humans

      This is a disingenuous argument. there are many things whose effects on humans can be fairly hypothesised without such experimentation.

      It seems that there is precedent and scientific merit to the expectation that a furin cleavage site between the S1 and S2 subunits of the spike proteins can enhance the fusion and hence infectivity of the holder of the spike protein.

    2. None of the staff at the Wuhan Institute were infected with SARS-CoV-2; they were PCR/antibody negative

      Isn't this rather suspicious that nobody from this institute happened to be infected as a part of the general transmission in the city?

    1. explain the puzzling fact that SARS2 has not changed since it first appeared in humans

      how substantial of a change are you considering? There have been many mutations in the spike protein which have gotten fixed in distinct sub-populations of variants in various regions/countries..

    1. Using the phoA gene and phoA fusions to monitor expression in these vectors, we show that the ratio of induction/repression can be 1,200-fold, compared with 50-fold for PTAC-based vectors

      with glucose?

    1. While the talks continue, Iran is keeping up the pressure by adding to its stockpile of highly enriched uranium and the equipment to make it, all in violation of the deal.

      That is an unfair statement, considering that context that the deal is no longer operational.

    1. cells are resuspended in ∼1.5 mL transformation storage buffer

      100 fold concentration from culture volume (150 ml) to final aliquots (1.5 ml) is a lot for chemical competent cells. For E. coli this is generally 10-20 fold only. I have only seen electrocompetent cells being that concentrated.

      This needs to be tried out and verified

    1. It is reported that functional metagenomic selections identify genes with less than 65% amino acid identity to known resistance genes

      How do you verify that these are actually causing resistance and not some native transporters etc. that pre-evolve resistance mechanisms?

    1. to study which conditions are necessary to generate interaction patterns like symbiosis or competition, and how higher order community structure can emerge from these

      By generation, you mean through evolution?

    1. These functions may arise from genomic scale differential expression and genetic polymorphism at the protein level, established throughout the microbiome/plant co-evolution and could explain its superiority over the maize resident plant-borne microbiome.

      Could the differential gene expression have something to do with being passaged on artificial culture media and other in vitro manipulations such as cryo-preservation?

      Repeating the same experiment but this time sampling the native maize microbiome into culture based collections and inoculating sterelized maize with this might explain a part of this?

    2. more than one-third of the total stalk bacterial endophytes grew specifically in one of the three culture media used, regardless of the type of media

      Is this a one off thing due to random variation or reproducible?

    3. A total of 17 wells containing highly abundant bacteria from the sugarcane core microbiome was selected to construct a synthetic community.

      Also curious to see the results on growth promotion of Maize, if all the wells are included (both high and low abundance bacteria)

    1. ssrA degradation tags depend on a short sequence on the N terminus of the protein for quick degradation by the ClpX system in E. coli

      Shouldn't it be C terminus?

    1. found that conjugation frequency curves strongly resemble bacterial growth curves, with a lag phase occurring after initial mixing of donor and recipient cells, followed by a period of increasing conjugation (e.g., an exponential phase) that typically ends in a plateau (e.g., a stationary phase)

      Does it depend on what growth phase the initial donor and recipients were taken from before mixing?

  4. Apr 2021
    1. The FDA clearance and a marked up media highlighting the popularity of the drug prior to the publication of its research could also be the reason why human cognitive biases could have added to the ‘placebo effect’ in a small number of patients

      This is a very interesting positive feedback loop. It is almost hypothesizing that the official approval or positive press of any intervention would increase the magnitude of the placebo effect. I wonder how this could be tested in a rigorous manner ... some kind of delayed arms in a trial?

    1. At the same time, many quorum-sensing systems are known to regulate traits that strongly depend on the local cell composition, like conjugative transfer6,7,8, which suggests that cells may profit from limiting their communication range to nearby cells.

      Conjugative transfer is one of the phenotypes of quorum sensing, there are many others which could be activated simultaneously. The argument says that conjugative transfer depends on the local cell composition, but why does that translate to cells profiting from limiting the communication range?

    2. This enables cells to accurately detect micron scale changes in the community composition

      This seems like an unsubstantiated phenotype being attributed to short range quorum sensing

    1. The solution is multilateral action in international institutions and international endeavors outside the WTO.

      I hate to say that this solution is very vague and needs to be elaborated

    2. undermining private IP rights would eliminate the incentives that inspire innovation, thus preventing the discovery and development of knowledge for new goods and services that the world needs

      Much of the discovery of knowledge is publicly funded research which has little to do with intellectual property rights and is not justified to be included in this statement.

    3. The primary justification for granting and protecting IP rights is that they are incentives for innovation, which is the main source for long‐​term economic growth and enhancements in the quality of human life

      Innovation for it's own sake when the products of such innovation are not reaching their intended consumers in order to alleviate the actual problem they were designed to solve seems rather pointless. Intellectual property rights were designed to encourage innovation, with the goal that such innovation would eventually be beneficial to society. Here there should always be a nuanced balance between the theoretical benefits of innovation and the actual benefits it is leading to on the ground. Hence any discussion of innovation should include the access and utility of such innovations to all stakeholders.

    4. practical reality of a world in which many medicines would simply not exist if it were not for the existence of IP rights and the protections they are afforded.

      There are always alternate incentives such as fixed cash awards, and subsidy transfers with fixed ceiling which could serve as incentive for innovation in such pandemic situations. It is not all or none

    5. have warned that allowing their COVID-19 vaccines to be copied without their permission through recourse to compulsory licensing “would undermine innovation and raise the risk of unsafe viruses

      Innovation is one thing but the safety of vaccines is for regulatory bodies to determine. Pharmaceutical companies have absolutely no say in this matter hence this is a straw man fallacy argument

    6. There is no evidence that intellectual property rights are a genuine barrier for accessibility of COVID‐​19‐​related medicines and technologies

      There are serious vaccine shortages in the developing countries, one key reason for which is that the developed countries have earmarked a lions share of the initial vaccine doses and due to limited manufacturing of the vaccine to satisfy the whole world's needs. Increased vaccine availability by enabling generic local manufacturers would enable much faster vaccination in all countries and providing essential doses for modest vaccination in the poorer countries

    1. Earthworms don’t do well in these hot temperatures compared to red wigglers which have a higher tolerance for temperature differences (they can survive at temperatures between 32 and 95°F / 0 - 35°C).

      Earthworms can survive 0-35C

    1. The similarity between pBBR1 and some plasmids of gram-positive bacteria has led us to precisely examine its mobilization function

      These similarities are all located in the RSA and the amino-terminal half of the pBBR1 Mob protein. Source: from the same paper below

    1. For many scientists, a new discovery is followed by a plan to make money, to form a company and get a patent.

      I doub't this assertion is valid.

    1. this expression contains two infix calls:

      Infix is where the function is not prefixed like usual f(.,.) so a <- b or a + b are infix calls

    1. , a hot-start PCR enzyme that prevents non-specific amplification from mispriming or primer-dimer formation during reaction mixture preparation

      So it doesn't prevent primer dimers forming during the reaction then? That is misleading marketing

    1. In contrast, SLiCE is an in vitro recombination method facilitated by bacterial cell extracts.

      What is the advantage of in vitro recombination by bacterial extracts as opposed to in vivo method?

      • Is the limitation the transformation efficiency of individual fragments?
    1. perhaps immunity will fade faster, for instance. But holding to the current dosing schedules means a slower vaccination program and more deaths.

      But going ahead to gamble with what could be an not sufficiently effective or temporary single dose without a line of sight for second dose could mean that once they discover and suggest people to take second doses, many people might lose trust and not show up for the second dose. This will be an irreversible effect on public trust on the vaccine

    1. when assembling Escherichia coli K12 MG1655. This genome can only be assembled into a single contig when the read length exceeds the size of the longest repeat in the genome, a multi-copy rDNA operon

      Demonstrates that if the read length exceeds the longest repeat, then automated assembly becomes straightforward

  5. Mar 2021
    1. When you have the data-variable in a function argument (i.e. an env-variable that holds a promise2), you need to embrace the argument by surrounding it in doubled braces, like filter(df, {{ var }})
    1. will show in real-time what downstream processes have been completed for each sample.

      This is impossible unless we upload data after each step. That would be too much work to expect from people who are busy in the wetlab

    1. Phanta Max Super-Fidelity DNA Polymerase is a new generation superior enzyme based on Pfu DNA Polymerase for robust PCR with extreme fidelity. High amplification efficiency and template adaptability makes Phanta Max suitable for almost all PCR reactions. The unique extension factor, specificity-promoting factors and plateau-inhibiting factor in Phanta Max greatly improve its long-fragment amplification ability, specificity and yield
    1. The enzyme offers fast amplification and strong strand displacement capabilities, making it ideal for nucleic acid amplification methods such as whole genome amplification, multiple displacement amplification and isothermal amplification.

      IsoFast™ Bst Polymerase

    1. patent protections and the profits they derive are a requirement for the innovation that yields lifesaving medicines.

      This is true. But if there are many lives that are not being saved, then what's the use of this innovation other than being a paper tiger?

    1. the kinetics of hybridization remain poorly understood, and no models or algorithms have been reported that accurately predict hybridization rate constants from sequence and reaction conditions (temperature and salinity). This knowledge deficiency has adversely impacted the research community by requiring either trial-and-error optimization of DNA primer and probe sequences for new genetic regions of interest, or brute-force use of thousands of DNA probes for target enrichment

      Why does kinetics impact the design of primer and probe sequences? Isn't it determined completely by thermodynamics?

    1. use of plasmid DNA enabled lower depth sequencing, and assemblies sufficient for full antimicrobial resistance gene annotation were obtained with as few as 2,000 to 5,000 reads, which could be acquired in 20 min of sequencing
    1. Make sure the 3′ end of the primer contains a C or G residue, because T and A residues bind more easily to DNA in a non-specific way

      This sounds wrong. G/C bind stronger hence provide for a better anchor for polymerase to start.

      G/C has more propensity for non specific binding though

    1. methylated motifs. These motifs often differ among species and strains24,25, making it possible to use combinations of methylated motifs (endogenous epigenetic barcode) for metagenomic binning.

      This is interesting. If methyl transferases are carried on mobile genetic elements, horizontally transferred to closely related organisms, shouldn't they share methylation signatures?

      Which would make the motifs not differ among species and strains?

    2. Our method takes advantage of these endogenous epigenetic barcodes to resolve individual reads and assembled contigs into species- and strain-level bins

      Do individual strains or closely related species vary so much in their methylation signatures?

    1. This work facilitates the reporting of promoters in absolute units, the variability in their activity across a population, and their quantitative toll on cellular resources

      Looks like this has been done in the past. Ido Golding's work already measures low numbers of copy number, mRNA counts simultaneously in phage infected E coli using microscopy + clever statistics

      • Wang, Mengyu, et al. "Measuring transcription at a single gene copy reveals hidden drivers of bacterial individuality." Nature microbiology 4.12 (2019): 2118-2127.nature
    1. getting severely ill when infected with TB if they inherited two copies of a rare variant of the immune gene TKY2, called P1104A.

      The article could have benefited from a short mechanistic explanation of the mechanism behind this

    1. Pointing to the country's priority list, announced way ahead of the vaccine rollout earlier this month, he said, "In the first seven to eight months, we are focused on the 30 crore people, about which we have talked quite often and we know who those people are, who are the needier".

      Is this an appeal for their votes?

    1. viXra will be open to anybody for both reading and submitting articles. We will not prevent anybody from submitting and will only reject articles in extreme cases of abuse, e.g. where the work may be vulgar, libellous, plagiaristic or dangerously misleading.

      What if ArXiv claimed they were also rejecting articles only in extreme cases. But since they don't have the staff to vet all the submissions they receive, they came up with the endorsement system.

      I don't see this new platform as solving any problem - this model will break when the scale of submissions increases

  6. Feb 2021
    1. Plate-reader measurements of bacterial growth in the presence of various antibiotic concentrations were used for MIC values. For the strains used in conjugative experiments (both within and across genera) as well as the post-conjugation transconjugants,

      Can the presence of the sweetness affect the MIC on any way? Can this be controlled for?

    1. He picked himself for the match so as to fulfill the BCCI criterion (which requires state administrators to have at least one first-class match experience) for becoming a selector at the state level. After the match, he appointed himself as the chairman of selectors of HPCA Ranji trophy cricket team

      Wow! smells like conflict of interest, ain't it?

    1. curcumin doesn’t have any of the basic qualifications of a good pharmaceutical. Studies have shown that rats absorb less than 1% of the curcumin they eat.

      This is a great argument if curcumin is being talked about as a pharmaceutical in the typical sense.

      But when consumed as a regular part of Indian cuisine, turmeric is generally added into hot oil along with other whole spices - this could possibly alter the bio availability of curcumin (as it could for fat soluble vitamins?)

    1. Urban Governance in India -- Episode 31 of The Seen and the Unseen (w Shruti Rajagopalan)

      Disconnect between power and accountability. Decentralization in politics

    1. A ribosome typically covers 10 codon positions and hence ℓ = 10 with the A-site of the ribosome located at the sixth codon position in this segment

      6th position from the downstream (3') side?

  7. Jan 2021
  8. www-pnas-org.ezproxy.rice.edu www-pnas-org.ezproxy.rice.edu
    1. Aminimumof%20basepairsinacompletelyhomologoussegmentisrequiredforsignificantrecombination.Thereisanexponentialincreaseinthefre-quencyofrecombinationwhenthelengthofhomologousDNAisincreasedfrom20basepairsto74basepairsandanapparentlylinearincreasewithlongerDNAsegments.Mis-matcheswithinahomologoussegmentcandramaticallyde-creasethefrequencyofrecombination.

      A minimum of 20 base pairs in a completely homologous segment is required for significant recombination. There is an exponential increase in the frequency of recombination when the length of homology increases from 20 to 74 bp and an apparently linear increase with longer DNA segments. Mismatches within a homologous segment can dramatically decrease the frequency of recombination.

    1. However, to our knowledge, there is no study that compares microbial communities in different CW designs with different wetland plant species treating pesticides.

      I would want to see a more persuasive reason for why the different designs need to be studied rather than just that no study has done it so far

    1. newly acquired spacers derived from pTrig were 34 times more prevalent in response to the signal than without, increasing from 0.038(±0.004)% to 1.28(±0.03)% among all arrays in the cell populations

      Dynamic range of 34 is much smaller than 400. It is interesting to understand what causes this.

    1. Dr Samiran Panda, a scientist at the Indian Council of Medical Research (ICMR), told The Wire Science earlier this week that the “mode” is effectively a single-arm clinical trial that wouldn’t have have a placebo and whose results wouldn’t be published in peer-reviewed journals – but with everything else being the same.

      Single arm clinical trials - good reference here Evans, Scott R. "Clinical trial structures." Journal of experimental stroke & translational medicine 3.1 (2010): 8.

    1. Key points from explainer video - Soil biodiversity conservation Youtube

      1. Need to be able to see the soil biodiversity (microscopic) - visuals
        • to get people excited
      2. Need data to figure out where this needs conservation
      • Above ground biodiversity is not correlated with below ground (!)
      • Need to harmonize protocols to collect this across the world
      • Along with biodiversity, we should measure function - how the biodiversity affects ecosystem functions
    1. This variant presents 14 non-synonymous mutations, 6 synonymous mutations and 3 deletions. The multiple mutations present in the viral RNA encoding for the spike protein (S) are of most concern, such as the deletion Δ69-70, deletion Δ144, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H
    1. ResCap capture library is a homemade core reference database (which will be available upon request) that comprises both well-known and hypothetical genes encoding resistance to antimicrobials

      Why is it not deposited along with this open-access paper? Isn't full data disclosure a part of the journal ethics?

    2. SeqCapEZ (NimbleGene) technology, which includes probes for 8667 canonical resistance genes (7963 antibiotic resistance genes and 704 genes conferring resistance to metals or biocides), and 2517 relaxase genes (plasmid markers) and 78,600 genes homologous to the previous identified targets (47,806 for antibiotics and 30,794 for biocides or metals)

      I wonder how the resistome is defined - does this also include single nucleotide variants that confer resistance? I assume it might be hard to capture such single nucleotide variants with probes

      For example of such variants Ramanathan, Babu, et al. "Next generation sequencing reveals the antibiotic resistant variants in the genome of Pseudomonas aeruginosa." PloS one 12.8 (2017): e0182524.

    1. the bacteria of the recipient strain was unable to grow on the plate containing ampicillin (> 100 mg/L) and gentamicin (> 50 mg/L) by drug sensitivity test

      Chlorine could affect the activity of the antibiotic ampicillin - was this tested for in the drug sensitivity test with various antibiotics?

    2. Non-lethal dose chlorine (0.5 mg/L) increased the conjugation transfer frequency,which confirmed that the mRNA expression levels of type IV secretion system (T4SS) proteins vir4D, vir5B and vir10B were significantly enhanced

      Does the fold change of increase in conjugation mirror the fold change of increase in mRNA expression of any of these genes?

      fig 4b -> conjugative transfer frequency increases by ~ 10 fold at 0.5 mg/l chlorine fig 4h -> genes increase only by 1.5 fold

      • on a side note, it is wonderful that the authors looked for a causal expression for increase in the conjugation. It is incredibly rare in these kinds of studies
    1. something that the alternative online media has been unable to do is where mainstream media platforms are still unrivalled – being a one-stop repository for different sets of news consumers. MSM digital platforms, despite many flaws, thrive as catch-all baskets for round-the-clock information seekers, ranging from politics to cricket, from fashion to music and important news (which doesn’t only mean the unusual massacres) from a hamlet in Arwal district in Bihar to that from a Delhi suburb.

      This will be impossible for a small digital media startup to achieve.

      I rather see the independent digital news as a gateway to important, contentious, controversial or rather plain and non flashy news. News of the kind ignored by mainstream media due to their perverse incentive structures.

      I don't think the coverage on cricket for example or entertainment from newslaundry would be any drastically different from mainstream media. In that sense it would be wiser not to compete and squander resources on such kind of reporting just to be a one stop shop.

    1. we developed a vastly improved INTEGRATE system that uses streamlined expression vectors to direct highly accurate insertions at ~100% efficiency effectively in a single orientation, independent of the cargo size, without requiring selection markers

      Efficiency sounds too exciting to not try!

    1. using a commercially available reagent (Bio-Rad SPR) that generates RT-qPCR-ready cell lysates with minimal manipulations. Similar reagents are now available from a number of sources (Ambion Cells-to-CT, Invitrogen CellsDirect, Roche RealTime Ready Cell Lysis, etc.)
    1. we demonstrate improved PCR-enhancing cocktails containing nonionic detergent, l-carnitine, d-(+)-trehalose, and heparin. These cocktails, in combination with two inhibitor-resistant Taq mutants, OmniTaq and Omni Klentaq, enabled efficient amplification of exogenous, endogenous, and high-GC content DNA targets directly from crude samples containing human plasma, serum, and whole blood without DNA purification.
    1. We found that a lysis solution containing both the non-ionic detergent (IGEPAL CA-630, chemically equivalent to Nonidet P-40 or NP-40) and bovine serum albumin (BSA) gave the best RT-qPCR yield. This direct lysis to reverse transcription protocol outperformed a column-based extraction method using a commercial kit.
    1. There are many commercial interests invested in persuading us to eat things that may be edible, but shouldn’t really be considered food

      :) very funny

    1. While no serious climate scientist doubts the fact that human activities are causing climate change, this can’t be proved through experimentation on another Earth.

      In both cases, the answers should be clear when looking at the evidence and the mechanisms at play without an ideological bias

  9. Dec 2020
    1. Fluorescence values for sfGFP (excitation, 485 nm; emission, 528 nm) and mCherry (excitation, 580 nm; emission, 610 nm)

      peak emission of sfGFP is 510 nm, why was 528 used here? fpbase

    2. . Some SsGCs exhibited universal activation across all hosts in both reporters (constructs A–C

      What do the sequence IDs (1-12) in this image correspond to the oligo ID?

    1. it was liable to impact the final results of the vaccine trial since, as more participants in the placebo arm would become infected, “the result of those getting the vaccine would obviously look better”.

      Wouldn't patients from both arms be impacted the same way by risky practices induced by the document?

    2. By giving the confidence to participants that they are safe after taking the shots, the participants may become complacent. The people in the placebo group, in reality, have got zero level of protection, hence increasing their chances of getting the infection,” he said.

      editor of the Indian Journal of Medical Ethics Amar Jesani

    1. it remains unclear exactly why resources are exchanged among trees in the first place

      Maybe focusing on the 'why' rather than the 'how' of a novel scientific study or paradigm prevents acceptance in the scientific community and slow down scientific progress?

    1. We recommend the following changes to the default settings when designing ddPCR assays:

      Primer3 : designing primers and probes for ddPCR

      In the General Settings window, change “Concentration of divalent cations” to 3.8, “Concentration of dNTPs” to 0.8, and “Mispriming/Repeat Library” to the correct organism ■In the Advanced Settings window, change both the “Table of thermodynamic parameters” and “Salt correction formula” to SantaLucia 1998 ■In the Internal Oligo window, we recommend setting 15 for the minimum number of bases for the oligo. We recommend 64°C as the minimum Tm for the probe, 65°C as the optimal Tm for the probe, and 70°C as the maximum Tm for the probe. These parameters can be relaxed to allow for smaller/larger oligos, which may be necessary for high GC or low GC targets. Oligo size should be no smaller than 13 and no larger than 30 nucleotides

      Note: After you have made the desired changes in Primer3Plus, select Save Settings under General Settings and save these parameters in a file. To apply these settings in the future, upload them by selecting Browse in the General Settings tab, find this file, and click Activate Settings.

    1. mRNA-1273 vaccine candidate, manufactured by Moderna, encodes the S-2P antigen, consisting of the SARS-CoV-2 glycoprotein with a transmembrane anchor and an intact S1–S2 cleavage site. S-2P is stabilized in its prefusion conformation by two consecutive proline substitutions at amino acid positions 986 and 987, at the top of the central helix in the S2 subunit
    1. Cell densities achieved with TB were over 3 times greater than those seen with the most commonly used broth, LB, and DNA yields were 2.5 fold higher

      high cell density => blocks spin columns?

      Have you observed that?

    2. In this study, we have quantitated yields of low copy and single copy number plasmid DNAs after growth of cells in four widely used broths (SB, SOC, TB, and 2xYT) and compared results to those obtained with LB, the most common E. coli cell growth medium

      TB (terrific broth) consistently generated the greatest amount of plasmid DNA, in agreement with its ability to produce higher cell titers. The superiority of TB was primarily due to its high levels of yeast extract (24 g/L) and was independent of glycerol, a unique component of this broth. Interestingly, simply preparing LB with similarly high levels of yeast extract (LB24 broth) resulted in plasmid yields that were equivalent to those of TB.

    1. ROI/Uniformity plate

      A regions of interest (ROI) calibration maps the positions of the wells on the sample block of the Applied Biosystems 7500/7500 Fast Real-Time PCR System. The 7500 software uses the ROI calibration data to associate increases in fluorescence during a run with specific wells of the plate. The instrument uses a set of optic filters to distinguish the fluorescence emissions gathered during runs. You must generate a calibration image for each individual filter to account for minor differences in the optical path.

      ABI 7500 manual:

    1. occupy the fill scale with a slightly darker version of the palette used for color.

      Using colorspace package.

      geom_boxplot(aes(color = season, fill = after_scale(desaturate(lighten(color, .6), .6))), size = 1)

    1. All the existing color palettes are available in Paletteer. Just specify the package and palette names to use!

      Ggplot, color palettes

    1. We report the preclinical development of BNT162b2, a lipid-nanoparticle (LNP) formulated N1-methyl-pseudouridine (m1Ψ) nucleoside-modified mRNA (modRNA) vaccine candidate that encodes P2 S with a native furin cleavage site resulting in the S1 and S2 cleavage fragments
    2. To generate the template for RNA synthesis, a DNA fragment encoding the SARS-CoV-2 P2 279 S protein (based on GenBank: MN908947), including the amino acid exchanges K986P and 280 V987P, was cloned into a starting plasmid vector with backbone sequence elements for 281 improved RNA stability and translational efficiency19,34

      BNT162b2

    1. Antibiotics, according to strain sensitivity, were used at the following concentrations: tetracycline 2 μg/ml for Rm1021, BM7, BM299 and BM300, 4 μg/ml for LMG18864, 10 μg/ml for other Sinorhizobium strains and for E. coli; streptomycin 600 μg/ml and kanamycin 200 μg/ml were used for Rm1021; kanamycin for E. coli S17.1 was used at 40 μg/ml; gentamicin was used at 20 μg/ml for all strains
    2. All Sinorhizobium strains were grown in TY medium (Beringer, 1974) at 30 °C

      TY contained, per litre, 5 g Difco Bacto- Tryptone, 3 g Difco Bacto-yeast extract and 1-3 g CaC1,.6H20.

      Source

    1. The slopes and efficiency for the SARS-CoV-2 standard curves were not consistent over time, confirming the need to include a standard curve each run rather than using a single curve for multiple plates.

      Is the standard curve performed with matrix or in pure water?

    1. Look at filmmaker Anubhav Sinha’s last three films. He went for Muslim minority story (Mulk) then he went to do a Dalit-related, anti-caste-related story in Article 15, and now he has gone for domestic violence (Thappad). He has touched religion, caste and gender.
    1. The voltage is kept low, ∼10 V/cm, where cm refers to the length of the gel

      This is wrong - the cm refers to the inter-electrode distance. It is a misonomer that people confuse it with the length of the gel

  10. gmo-crl.jrc.ec.europa.eu gmo-crl.jrc.ec.europa.eu
    1. Since most of the power produced in the electrophoretic process is dissipated as heat the following detrimental effects can result: • an increased rate of diffusion of sample and buffer ions leading to broadening of the separated samples • the formation of convection currents, which leads to mixing of separated samples; • thermal instability of samples that are rather sensitive to heat (e.g. denaturation of DNA) • a decrease of buffer viscosity hence a reduction in the resistance of the medium
    1. found that the P R promoter from rhizobial phage 16-3 was active in M. extorquens and engineered the promoter to be inducible by either p-isopropyl benzoate (cumate) or anhydrotetracycline. These hybrid promoters, P R/cmtO and P R/tetO, were found to have high levels of expression in M. extorquens with a regulatory range of 10-fold and 30-fold, respectively
    1. Currently, the majority of quorum sensing systems used in synthetic biology rely on self-produced small molecules that result in spatially and temporally self-organized systems, which can not be easily externally regulated

      Why can't the LasI or LuxI proteins be inducible?

    1. appropriately selecting sets of functionally redundant species and adding their abundances can help identify environmental drivers of microbiome composition

      The set of functionally redundant species will be specific to the environmental parameter in question and members could possibly be present in multiple functional groups corresponding to different parameters also.

      For example: When looking at nitrate level, species A,B,C are the same functional group, for sulphate species B, D, E are same group, for early colonizers A, D, F etc.

  11. www.jstage.jst.go.jp www.jstage.jst.go.jp
    1. Since currentanalytical technologies for genes and metabolites provideonly a “snapshot” information at the time of measurement,temporal shift s in plan t an d microbial physiologies hav e bee na majo r obstacl e to detaile d analyses. Therefore, a stabl e syn‐thetic system may facilitate clearer investigations of plant-microbe interactions, leading to unique insights in this field

      snapshot vs dynamic information

  12. Nov 2020
    1. Here we present a detailed, genome-wide transcriptomics and proteomics dataset of E. coli grown under 34 different conditions. Additionally, we provide measurements of doubling times and in-vivo metabolic fluxes through the central carbon metabolism

      34 different conditions:

      We manipulate concentrations of sodium and magnesium in the growth media, and we consider four different carbon sources glucose, gluconate, lactate, and glycerol. Moreover, samples are taken both in exponential and stationary phase, and we include two extensive time-courses, with multiple samples taken between 3 hours and 2 weeks

    1. But humans, we have demonstrated, are not inherently self-interested.

      Would be good to build a tighter argument for this demonstration before writing such a strong statement

    2. some anthropologists now argue that millions of years of evolution have hard-wired us for altruism, not self-interest.

      Altruism has also been difficult to explain, even in more primitive organisms such as microbial communities which have much simpler behaviors.

      Isn't it a stretch to claim that it is hard-wired through evolution by giving specific examples of altruism in primitive humans and primates?

    1. Since each strand is replicated independently, it is possible for the ssDNA form to accumulate.

      How do you deal with ssDNA during plasmid extraction?

    1. Previously, we and others have shown that the inclusion of an antitoxin and the careful balancing of toxin and antitoxin expression levels across relevant conditions can mitigate this effect, resulting in a kill switch that is evolutionarily stable over biologically relevant periods
    1. Cells are aliquoted into chilled tubes, frozen in a dry ice bath and stored at −80 °C until use.

      How long do the cells hold their competency?

    1. Solutions can be autoclaved for 15-20 minutes at a maximum of 121 °C. There will be some hydrolysis to glucose and fructose, depending in part on how rapidly the autoclave comes to the required temperature and pressure. Care must be taken to prevent solutions from caramelizing.
    1. Antibiotic concentrations used for plasmid selection in V. natriegens: ampicillin–carbenicillin 100 µg ml−1, kanamycin 75 µg ml−1, chloramphenicol 5 µg ml−1, spectinomycin 100 µg ml−1. E. coli experiments were performed in standard LB and M9 media.
    1. A moving average is commonly used with time series data to smooth out short-term fluctuations and highlight longer-term trends or cycles. The threshold between short-term and long-term depends on the application, and the parameters of the moving average will be set accordingly.
    1. partial amplification of the dcp gene, located in close proximity to terC, opposite the oriC region

      TerC: qPCR copy number of e coli chromosome

    1. Replication begins

      Rolling circle replication overview

      Replication begins when the Rep protein, which is encoded on the plasmid (ORF A), recognizes a specific site on the plasmid (double-strand origin, or DSO) and catalyzes the nicking of one DNA strand. The Rep protein remains bound to the 5′ phosphate after the nicking action. The newly released 3′ hydroxyl on the opposite end serves as a primer for DNA synthesis. The host DNA polymerase uses the unnicked circular strand as a template, so that a single replication fork moves around a plasmid until it regenerates the DSO. A second copy of Rep protein catalyzes the cleavage of the newly formed DSO, effectively releasing a single stranded copy of the plasmid. In the absence of Rep, the replication fork continues to move around the template, forming a single stranded concatemer. The single strand origin (SSO), a non-coding element that forms extensive secondary structure, is required for synthesis of the lagging strand. SSO sequences vary considerably among different RCR plasmids, but are extremely important for robust replication of the plasmid in the cell [10]. Here we describe the engineering of the pWV01 RCR origin to create pBAV1K-T5, a very broad-host range expression vector.

    1. Designed probes were modified at the 5′ end with the fluorophore FAM for the reference gene cysG and HEX for the plasmid marker oriT, and furthermore modified at the 3′ end with the quencher BHQ-1

      plasmid copy number qPCR assay.

      Why cysG: Siroheme synthase gene?