1,462 Matching Annotations
  1. Last 7 days
    1. There are a number of ways to include dynamic variables within documents rendered by Quarto

      Can this work within the yaml header as well? - Would be relevant to the question raised by this thread

    1. This preparation includes fragments from the RNA-dependent RNA polymerase and VP1 (ORF1-ORF2 junction) regions.

      of which strain of Norovirus?

    1. (Cog1F and Cog1R for GI viruses; Cog2F and Cog2R for GII viruses) (27), and 200 nM each probe (Ring 1E, FAM-TGG ACA GGR GAY CGC-MGBNFQ, where FAM is 6-carboxyfluorescein and MGBNFQ is minor groove binder and nonfluorescent quencher

      Ring 1E requires MGB for best performance since it has a low Tm otherwise

    1. Callouts are an excellent way to draw extra attention to certain concepts, or to more clearly indicate that certain content is supplemental or applicable to only some scenarios.
    1. This item type can also be adapted to fit many types of unusual items.

      report

    1. When a desired DNA is present at low concentrations relative to nearly identical undesired DNAs, LNAs can block amplification of unwanted DNAs

      I don’t understand what they mean by blocker? Doesn’t the stuff also get extended like primers or cleaved like probes?

    1. Ensuring that each amplicon is only copied by its “correct” SuperSelective primer
    2. shorter foot lengths, though lowering the equilibrium abundance of foot hybrids, resulting in longer delays before the threshold cycle is achieved, lead to enhanced selectivity

      Why don't you show selectivity directly with the difference in Cq for same concentration of target and non-target sequence?

    1. Although single-color ddPCR assays have been already utilized to assess SNVs at low VAFs24, it is required to design two different allele-specific primers to detect one SNV (wild-type and mutation-specific assays)

      The two different allele-specific primers are required to compete with each other?

  2. Mar 2024
    1. All reference sequences (ABCC9 and SNV wt assays) were located in copy-number-neutral regions. Sequence details are available upon request.

      Why won't you just put them here?

    1. SuperSelective primers, which enable the amplification of SNVs in the presence of an excess of the corresponding wild-type target

      See Fig 1A in ref 13

      Vargas, D. Y., Kramer, F. R., Tyagi, S. & Marras, S. A. E. Multiplex real-time PCR assays that measure the abundance of extremely rare mutations associated with cancer. PLoS ONE 11, e0156546 (2016).

      imgur

    1. SNP PI3Kca_E545K

      How many nucleotides is this?

    2. An RMD assay comprises a single set of primers plus two competitive probes (each probe with a different fluorophore), one detecting the wild-type allele, and one detecting the variant allele.

      RMD = Rare mutant detection

    3. Drop Off (DOF) Determines absolute quantification of targets for assays designed to detect non-wild type sequences, such as indels and genome edits. The experiment type isdesigned to support an assay strategy where one probe counts all alleles andone “drop-off” probe sits on top of a predicted cut site.

      Is this better/more robust than detecting each allele with a different probe? - ie) using direct quantification of individual variants?

    4. Tm enhancers for probes are recommended for single nucleotide polymorphism (SNP) and rare mutation detection assays in order to keep the background fluorescence to a minimum. Shorter probes discriminate better between single base differences in the target amplicon(s)

      Does higher T annealing mean lower background? Or is there something else that relate Tm enhancers to background fluor - Could be to improve discrimination between the WT and the SNP variant?

    1. gate for only particles of bacterial size

      How do you determined where the "bacterial" sized particles should appear?

      Set a gate for bacterial size on a bivariate SSC-A vs FSC-A plot for events of bacterial size by including the donor strain and excluding all events caused by a sterile pyrophosphate buffer control. Source: Klumper, 2018 Spring protocol handbook

    1. Relative expression level measured by RNA QASeq in four clinical FFPE tumor tissue samples and three normal placenta FFPE samples

      LS : how are they able to detect 3 orders of magnitude difference? Won't the high abundance one hog most of the reads? - I guess the read depth should be 10 to 100x higher than the range you want to span?

    2. 10 ng Human PBMC gDNA from the same healthy donor was used per experiment, corresponding to 2,790 haploid copies

      How was this determined?

    1. Windows 10 has a built-in calibration tool that you can use to fix various display issues associated with your monitor.

      the one called "calibrate display colour" doesn't do this?

    2. experiment with different screen resolutions within Windows 10 Settings until you get to one that looks the way you want.

      This is hard.. most other of the resolutions in the drop down list give you blank screen space at the sides or too large content. - There needs to be a customization of the resolution to arbitrary values while maintaining the aspect ratio. I assume something like this would be possible in a linux system through the commandline?

    1. You need to turn off overscan, either on the TV or in the graphics card settings.

      Look for overscan in settings Question

      Sharp Aquos 26D44 connected to my new computer via a VGA cable and it displays fine. However, now that I've tried it with a DVI to HDMI cable it cuts off the edges of the desktop

    1. or changes between two files on disk

      I assume they have to be in the same repo?

    1. HTML Options

      This was not intuitive but these options go under this and this Posit community page was very helpful format: html: ..

  3. Feb 2024
    1. This approach exploits the equivalence between confidence intervals and hypothesis tests, detailed in Section 12.2.
    2. we must add aes(y = ..density..)) to the call to geom_histogram, which rescales the histogram to have area 1 (just like a density function has). We can then add the density function using geom_function

      See note for current version from stackoverflow

      The dot-dot notation (..density..) was deprecated in ggplot2 3.4.0. Please use after_stat(density) instead for the aes(y = )

    1. The data on the 463 courses at UT Austin can be found in the evals data frame included in the moderndive package
    1. a digestion with Plasmid-Safe DNase (Lucigen, Madison, WI, USA) was performed to purify the extracted plasmid DNA

      Digest unwanted contaminating linear double-stranded DNA, without harming circular double-stranded DNA such as plasmids and fosmids. LGC Biosearchtech

    1. Incremental reveal Not limited to just bullet points... Use `. . .` syntax to indicate a break at an arbitrary location . . . ```{r} head(mtcars) ```

      quarto incremental reveal

    1. To start and end a video, you need to use ?start=<seconds> and ?end=<seconds> to define the starting and ending times. Something like this:https://www.youtube.com/embed/xxx6x67ws7?start=45&end=200

      Youtube, url with start and end

    1. 10,000 units

      100,00 U/ 200 U/ul = 50 ul => 50 reactions using 200 units = 1 ul/20 ul rxn.

    1. Any other educational assistance that is excludable from gross income (tax free),

      Can this be considered a definition of tax free?

    2. Your filing status is married filing separately.

      Cannot claim credit

    3. credits are based on the amount of adjusted qualified education expenses paid for the student in 2023 for academic periods beginning in 2023 or beginning in the first 3 months of 2024.

      paid FOR the student?

      Paid by whom if not the student themself?

    4. Qualified education expenses paid on behalf of the student by someone other than the student (such as a relative) are treated as paid by the student.

      Does Tuition paid for graduate students by the professor (from research grants) count as a third party?

    1. Scholarship - partially taxable. Amount spent on tuition and qualified education expenses (provide link) not taxable, the remainder is taxable ordinary income. Reported on 1098-T if from USA stipend is treated as a Scholarships
    1. You cannot claim a credit for education expenses paid with tax-free funds. You must reduce the amount of expenses paid with tax-free grants, scholarships and fellowships and other tax-free education help.

      What are tax-free funds? Are fellowship / tuition for grad students paid by faculty grants counted as tax free?

    1. a formal billing arrangement is an arrangement in which the institution: (1) bills only an employer or a governmental entity for education that the institution furnishes to a student

      Applicable for graduate students

    1. Asking questions

      add caveat on timing if you need urgent answer:

      Please note that when posting a question on the discussion board, the TA's will not jump in until 24 hours after posting to give others a chance to answer and earn credit. If you need an immediate answer, put an Urgent tag on your question and one of us will email you the response in a few hours.

    1. git log --oneline --graph --decorate --all

      show commits of git as a tree

      To show the last 2 commits use this! git log --oneline --graph --decorate --all HEAD~2..HEAD

    2. git config --global alias.tree 'log --oneline --graph --decorate --all'

      configuration to make a shortcut to show commits of git as a tree

    1. I use Bash shell .sh scripts in my Windows projects to automate Infrastructure as Code, then run these scripts using Windows Subsystem for Linux - WSL I need to make sure the line endings of my Bash scripts are in the Unix style LF or \n and not Windows CFLF or \r\n

      This is different from the typical use case within windows, for example using git bash

    1. . The gh-pages branch method is based on an old GitHub Pages publishing option. This branch is somewhat separate from the rest of the version control tree

      This might be useful to access/look at the rendered website as files for troubleshooting purposes

    1. 250 μl of sample was loaded into the first well of each row in a 96-well plate, and 10-fold serial dilutions were made using a multichannel pipette (Rainin, Emeryville, CA) by transferring 20 μl from column i into 180 μl of medium in column {i+1}, mixing 10 times, and repeating the process; pipette tips were changed between dilutions

      How important is changing tips between dilutions?

    1. However, none of these approaches combines the simplicity, low cost, dynamic range and versatility of simply diluting cells and then growing drops on solid media as first proposed in ref. 17.

      First reference to CFU method from 1938!?

    1. Pipet 100 μL of Starting Culture into Dilution 1. Discard tip. Do NOT pipette up and down. Vortex tube for 5-10 secs

      serial dilution method. Why should you not pipette up and down? Cells killed due to pipetting stress?

    1. The problem raised by the #barbarplot campaign is that bar plots are a poor summary of the distribution of data.

      This is when data is very far from being normally distributed right?

  4. Jan 2024
    1. a kanamycin resistance gene known to be expressed in both Gram-negative and Gram-positive bacteria

      aphA-3 gene from Campylobacter coli. Ref 21, 25 ~ quoted

      A 1427-bp DNA fragment containing the kanamycin resistance gene, aphA-3, of plasmid pIP1433 from Campylobacter coli was inserted into a shuttle vector. Full expression of aphA-3 was obtained in Bacillus subtilis and in Escherichia coli.

    1. generally derives from variations in filtering out spurious and low-abundant sequences (e.g. Edgar, 2017; Prodan et al., 2020).

      DADA2 like ASV vs OTU?

      Applying different workflows on the same data will always demonstrate a certain level of variation among pipelines. These variations are usually most obvious in terms of the reported number of features.

    1. Since less alcohol is required for isopropanol precipitation, this is the preferred method for precipitating DNA from large volumes. In addition, isopropanol precipitation can be performed at room temperature, which minimizes co precipitation of salt that interferes with downstream applications
    1. Summary of the experimental setup

      Is there supplementary data in an intact mouse microbiome (without initial streptomycin treatment) for true in situ editing?

    2. Dashed line: Background fluorescence of cells before transduction.

      why not do a background subtraction?

    3. the P2-STF chimera encoded on a plasmid (p938)

      Why not the prophage genome?

    4. tail tip protein gpJ recognizes the LamB outer membrane porin and results in an irreversible binding of the phage to the cell surface

      Why irreversible? Covalent bond?

    5. Efficient conjugative delivery to different strains and species will thus likely require different specialized systems

      The same is true for phages as well

    1. Full length Illumina tagged primers used in study:

      27F (AGMGTTYGATYMTGGCTCAG) and ?R primer (GCTGCCTCCCGTAGGAGT) used

    Annotators

    1. using the FACS tosort for all three gates at highest speed by sorting for gain

      Note 19

      To be able to perform an efficient second purification sort, at least 20,000 presumptive transconjugants should be sorted in the first sort to account for a sufficient concentration of cells for the second run.

    Annotators

    1. why should researchers make websites? Because it is the easiest way to disseminate your work, your projects, who you are as a researcher, and to have more impact and outreach.

      Researchers should make websites to disseminate information better than journal publications

    1. One of the most useful features of Quarto reveal.js slides is absolute position, which allows you to specifically place elements on a slide.

      How do you get the exact position other than by iterative testing which is a pain due to the long render time of quarto (and no quick rendering such as infinite_moon_reader() that xaringan slides have

    1. using the FastStart High Fidelity PCR System

      Source: Sigma/Roche

      The FastStart™ High Fidelity PCR System is a blend of FastStart Taq DNA Polymerase and a thermostable proofreading protein, which is also chemically modified. This protein mediates proofreading activity, but carries no polymerase activity. Both proteins are inactive below +75 °C and are activated by heating to +95 °C for two minutes.

    1. 515F GTGCCAGCMGCCGCGGTAA Universal Turner et al. 1999

      Turner et al 1999 paper only worked with cyanobacteria right? How is this universal then?

    1. Fluorescence Activated Cell Sorting (FACS).  From sorting cells for single cell genomics to identifying the host range of a plasmid; from detecting metabolically active cells in an environmental sample to studying colitogenic-driving intestinal bacteria, FACS can be used in a multitude of applications to study microbial communities (Rinke et al. 2014, Klumper et al. 2015, Kalyuzhnaya et al. 2008, Hatzenpichler et al. 2016, Palm et al. 2014).
    1. zygotically inducible

      Interesting choice of terms!

    2. we report on a new approach to quantify the fraction of a bacterial community that is able to receive and maintain an exogenous conjugal plasmid termed community permissiveness

      How is this term different from conjugation frequency?

    1. This pool best represents the cells that had the potential to receive the plasmid, compared with the initial wastewater community before mating, i.e. before the few rounds of division that occur on the filter during incubation

      Interesting. This accounts for any bias in growth on the filter + nutrient media

    1. Conjugation events were visualized by stereomicroscopy and quantified by automated image analysis

      Why didn't you use flow cytometry data for this?

    2. Filters were incubated for 48 h at 25 °C, before 72 h storage at 4 °C for GFP maturation.

      Why that long for GFP maturation?

    1. a shows the sorting of the initial soil bacterial recipient community

      fig2a/gate II and III show that red has much higher autofluorescence than green in soil recipient community!? - Gains could also be quite different contributing to this?

    2. (d) shows the enrichment of transconjugants after the first fast enrichment sorting step to over 80% transconjugal cells

      carryover seen in - - donor in II left - Soil particles in III left (and right too)?

    1. This model allows for the lessons to remain free from brand integrations and sponsor messages, without introducing paywalls.

      Donation model

    1. I wash and dry my pan immediately after each use, but it’s what happens next that matters: I heat it gently over a low flame and add a bit of ghee. Then, I grab my dedicated “cast iron cloth” and rub the oil into the bottom and sides of the pan. I wait until the pan feels warm, but the ghee isn’t shimmering and snapping. Then I simply turn off the heat and let the pan cool naturally. This small step keeps it glossy and well-seasoned.
    1. To save a copy of the slide with your drawings, your best option is to print your presentation from the browser.

      Would be nice to save the scribbles as a .png with named with which slide the drawing was on..

  5. Dec 2023
    1. When creating line art, please use the following guidelines:

      what is line art?

      Line art or line drawing is any image that consists of distinct straight lines or curves placed against a background, without gradations in shade or hue to represent two-dimensional or three-dimensional objects. Wikipedia

    2. White space must be cropped from the image, and excess space between panel labels and the image must be eliminated.

      how much is excess?

    1. The blastn parameters used for VecScreen are significantly more stringent than the default blastn parameters

      The VecScreen parameters are pre-set using blastn options: -task blastn -reward 1 -penalty -5 -gapopen 3 -gapextend 3 -dust yes -soft_masking true -evalue 700 -searchsp 1750000000000

    1. what economists call rents: that is, value extracted through the ownership of a limited resource

      How does one draw the line between rent for providing a useful platform and extractive rent seeking?

      For example, does it help if the platform undergoes regular improvements, helps in information transparency with better reviewing systems, takes user feedback (both seller and customer)?

    2. Any abuse of market power is likely to show up first on the supply side

      Monopsony?

    3. The marketplace is designed and controlled by its owners, and that design shapes “who gets what and why” (to use the marvelous phrase from Alvin E. Roth, who received a Nobel prize in economics for his foundational work in the field of market design.

      This sentence is equally applicable even for overall markets (yes, even "free" markets)

    4. control the market through the algorithms and design features that decide which products users will see and be able to choose from. And these choices are not always in consumers’ best interests.

      digital platforms and marketplaces

    5. Likewise, Google does not just compete with other search engines like Bing and DuckDuckGo, but with everyone who produces content on the world wide web. Apple’s iPhone and Google’s Android don’t just compete with each other as smartphone platforms, but also with the app vendors who rely on smartphones to sell their products.

      This is slightly different from the direct competition Amazon has due to its own product line. Google search and Google play don't really generate their own content or apps that "compete" with others in their platform.

      Rather than competition this needs a different word that suggests the preferred placement that marketplaces and platforms can use to generate revenue.

    1. If this is better than mash, I wonder why it didn't take off? - It has only 40 citations compared to 2,000 of mash (as on Dec, 2023)

    1. To do so, we prepared 96-well plates with two-fold dilutions of each antibiotic, ranging from 400–6.25 µg/ml, in 100 µl of medium per well

      Does the liquid MIC extrapolate well to agar?

    1. However, Google Docs has several limitations (like not automatically doing sequentially number headings - a plugin to do this available but is buggy) so I would like to do the final polishing in Word

      google docs vs word in context of zotero

      I am working on a chapter collaboratively in Google Docs and the Zotero plugin worked like a charm for this

    1. Download the pre-sketched RefSeq archive (reads not provided here; 10x-100x coverage of a single genome with anysequencing technology should work):refseq.genomes.k21.s1000.msh
    1. P. lundensis is a Gram-negative, polar flagellated bacterium belonging to the Pseudomonas fragi clade of the Pseudomonas fluorescens species complex

      figure 2 from ref [7]

    1. sample size must be between 3 and 5,000 in order to use the shapiro.test() function.

      Does a sample size 3 really work?

    1. RNA guides and corresponding target RNA, ranging from 25 to 373 nt, present signals

      This screen needs to be repeated with the U64 design splicing into the 16S - Since 16S is inherently structured, it might be the cause for less complementation compared to the gfp:gfp splicing - My hypothesis: longer guide region would be better for the 16S designs since they can shift the 16S-ribozyme base paired construct to be more stable than 16S itself - This can be tested with a custom interaction sequence that is structured in different levels in the gfp:gfp splicing assay of 2B

    1. Characterization of 5 RENDR designs split at identified split sites.

      What splice site from Fig 2 is used in the RAM (Ribozyme addressable memory) paper now? - Answer : (15 : IGS/6 bp + P1/9 bp)

    1. You can also create human-readable block identifiers by adding a blank space followed by the identifier, for example ^quote-of-the-day, at the end of a block:

      If it's a bullet point with sub-points, the ^quote-.. needs to be at the end of the last sub-bullet point

    1. When subjects are instructed togenerate a random sequence of hypotheticaltosses of a fair coin

      for example, they produce sequences where the proportion of heads in any short segment stays far closer to 0.5 than the laws of chance would predict (Tune, 1964) // Thus, each segment of the response sequence is highly representative of the "fairness" of the coin

      • Could this be a nice idea to have student predict 10 consecutive coin toss outcomes and compare to a simulation?
      • Explain concepts of probability, intuition of "representative" sampling

      Subjects act as if every segment of the random sequence must reflect the true proportion: if the sequence has strayed from the population proportion, a corrective bias in the other direction is expected. This has beenc alled the gambler's fallacy.

    1. Why is statistics difficult!? - Good example to separate immediately measured tangible variables and imaginary/statistical properties such as probability

    1. dadasnake wrapper eases DADA2 use and deployment on computing clusters without the overhead of larger pipelines with DADA2 such as QIIME 2

      [dadasnake vs Qiime2] on clusters

    1. mapping these reads back onto high-quality OTUs/ASVs if matching at ≥ 97% sequence identity

      97% identity is not compatible with the Amplicon Sequence Variant approach which requires 100% identity right? - Might these mid quality reads be incorporated by re-running DADA2 but giving more weightage to the high quality ASVs / re-using their error profiles?

    1. In contrast, it is the removal of rare taxa that would appear to remove valid data.

      There is no clear consensus on if rare taxa are actually valid or not.

    1. mRNAs triphosphorylated at their 5′ ends.

      Good illustration of the 5' triphosphate in Wikipedia - 5' cap structure ; image

    1. However, few examples of such multi-level regulation have been implemented to date20,21. This has resulted in an unclear picture of how best stringent multi-level control can be achieved and the trade-offs that exist between performance, regulatory complexity, and cellular burden when designing these systems.

      Would have loved to see a better discussion of results from 20 which seems to have a nice emphasis on reducing leak and testing it with highly sensitive protease activity + western blot method

    1. The LasR sensing module includes the gene encoding LasR (BBa_C0078)

      This is wrong part #. It should be BBa_C0179

      C0078 encodes LasI - produces C12 AHL (AI-1)

    1. Others argue for a more ecumenical approach, encouraging researchers to try multiple methods on the same data set.

      This is a disaster of a recommendation and will cause the average scientist to present the method that gave the most favorable conclusions rather than be "more creative and find out why different methods gave different answers"

    1. or replicates, no statistics should be shown, because they give only an indication of the fidelity with which the replicates were created: they might indicate how good the pipetting was, but they have no bearing on the hypothesis being tested6.

      Talking about technical replicates You are advocating for throwing out technical variation and showing only the mean, isn't this misleading as to any issues with reproducibility in the experimental techniques that readers can anticipate?

    1. errors would reflect the accuracy of pipetting, not the reproduciblity of the differences between the experimental cells and the control cells

      It depends on where you believe the differences between replicates are arising from? - When making the mutations - when growing the cells with the mutations

    1. chose the introduction because this section of a paper is fairly easy for ChatGPT to write if it has access to background literature

      Pros and cons of having chatGPT write the introduction - Saves time for researchers ; without too bad of a job - Erodes a vital chance for researchers to deep dive into literature with the deliberate intention of writing - could be helpful in generating new ideas

  6. Nov 2023
    1. rigid pili, such as Inc N, P and W, conjugate at rates 2–4 orders of magnitude higher on solid surfaces
    2. flexible pili (e.g. Inc F, H and I), transfer equally well in liquid as in solid surfaces
    1. synthetic arrays of regulatory RNA could be created that achieved our design goals

      recombinogenic in other organisms?

    1. these commonly used artifacts are small code snippets that are entirely functional in nature and, therefore, when used in isolation, don't enjoy copyright protection at all.

      They are not "used" in isolation though, as it is the context of these code snippets gleaned from tons of code that makes it valuable to the ML scene in comparison to static autocompletion of the prior generation

    1. The donor and recipient populations grow according to the following standard exponential growth equations

      Includes no death rates and carrying capacity. Under what circumstances would this assumption be valid? - if the duration of conjugation (t) is well within the exponential growth phase maybe?

    1. The primary color palette uses three colors. Grey and white should be employed liberally ashigh-contrast elements. Red should be used sparingly to label and decorate symbols and information.We use this pink/red because it is more gender-neutral than the typically masucline color palettes ofother space/geospatial-focused groups and it is distinct from the green/earth tones of conservationgroups. It’s also effective for drawing attention to specific elements

      minimalistic colour palette

    1. apparent permissiveness (AP). It is defined as the ratio of the relative abundance of an ASV in the transconjugant pool to the corresponding recipient pool

      How is this different from conjugation efficiency?

    1. triggering homologous recombination and creating genetic instability10,11,12,13
    2. We applied machine learning to explain how specific interactions controlled the promoters’ transcription rates.

      Is explain the correct word here? - Maybe "fit"?

    1. Wilcoxon rank sum tests were used to test for significant differences between two groups

      Why using non parametric?

    1. Each template molecule in a sample is tagged with a UMI sequence consisting of 10–20 random bases, which can subsequently be used to sort and analyze reads based on their original template molecule

      Key logic that increases accuracy - You are sequencing the same template with the same UMI multiple times (15x here for nanopore) (since it is amplified a bit after the UMI is attached) - Since errors are random, you can bin the molecules with the same UMI (errors in the UMI can be reconciled since the sequences are mapped to the known reference) - Errors are fixed by generating a consensus of things in the same bin - The final UMI consensus error rate is < 0.01% down from 1% (?) in nanopore's native R10.3 technology

    1. Three distinct rRNA sequences exist in the operon, encoding for the two subunits of the ribosome; the 5S and 23S sequences code for the large subunit, while the 16S sequence encodes for the small subunit.

      rRNA operon

    1. demonstrate that targeting of 16S variable regions with short-read sequencing platforms cannot achieve the taxonomic resolution afforded by sequencing the entire (~1500 bp) gene

      Case for nanopore of entire 16S gene (1.5 kb) for better taxonomic resolution

    1. New methods are needed that can manage and help organize this scale of data. To address this, we consider the general problem of computing an approximate distance between two sequences

      the distance is required to search a database for closest entry to a query

    1. we inferred the parameters of a linear function fit to the log transformed gDNA concentration versus detection time (Fig. 2d–g). The inferred slope of the linear function is determined by the cell doubling time (~0.5 h) and intercept is determined by the background mutation frequency

      Interesting.. so it was not from least squares fitting to the data then..

    2. gDNA concentration versus the detection time for the (d) EC sensor, (e) ST sensor, (f) SA sensor and (g) CD sensor

      what is driving these differences? the homology sequence influences the kinetics of recombination?

    3. A threshold of GFP fluorescence 400 arbitrary units (a.u.) was used to determine the detection time for each gDNA concentration.

      what was the basis for this threshold?

    4. expressed from the IPTG-inducible promoter Phyperspank

      what promoter is this? - Bacillus promoter from IGEM, paris 2011

    5. cell-based DNA detection is relatively simple and cost-effective. In comparison, other DNA detection methods such as next-generation sequencing and quantitative polymerase chain reaction require specialized instruments

      Is it simpler to sit and count colonies vs doing qPCR? It is probably cheaper..

    6. Next-generation engineered bacteria hold tremendous promise

      What is specifically next generation? when does this begin?

    1. ubstantial contributions to conception anddesign, or acquisition of data, or analysis andinterpretation of data;

      acquisition of data is an ambiguous phrase here. What if somebody is involved in a preparative protocol where materials are produced but not the acquisition of data?

    1. Your questions should be reasonably scoped. If you can imagine an entire book that answers your question, you’re asking too much.

      hahaha, very useful statement!

  7. Oct 2023
    1. A method was developed for oriented immobilization of bacteriophage T4 through introduction of specific binding ligands into the phage head using a phage display technique

      Why was this method developed?

    1. UMAP embedding of Record-seq data

      Why UMAP for some (4B) and PCA for other dataset (4E)?

    2. metabolites and RNA are short-lived, omics-based measurements of transient stimuli only yield a snapshot of highly dynamic processes.

      Examples of transient stimuli in other context? - In this case, flowing through the intestine with changing conditions makes things transient.

    1. Lactococcal strains were routinely grown in M17 medium (49) supplemented with 1% glucose (GM17 medium) or, when needed, in chemically defined SA medium (26) at 30°C without shaking

      why without shaking?

    1. Acinetobacter baylyi ADP1 (30) and ADP1-ISx (11) were grown at 30°C in LB-Miller (10 g NaCl, 10 g tryptone and 5 g yeast extract per liter) or ABMS minimal medium (40)

      Reference for ADP1-ISx strain

      1. Suárez G.A., Renda B.A., Dasgupta A., Barrick J.E.. Reduced mutation rate and increased transformability of transposon-free Acinetobacter baylyi ADP1-ISx. Appl. Environ. Microbiol. 2017; 83:e01025-17. [PMC free article] [PubMed] [Google Scholar] [Ref list]
    1. a biologically driven cryptic cycle was determined by identifying metabolically active sulfate reducing and sulfur oxidizing lineages co-locating within the sediments, effectively masking sulfide production through re-oxidation back to sulfate.
    1. Corynebacteriumglutamicum

      C. glutamicum is a gram positive of phylum actinomycetatota - 24/Oct/2023

    1. typically between 1010 and 1011 microbial cells per wet-weight gram of faeces7,8,9.
    2. This figure is commonly mentioned in the microbiome literature, but its source has been difficult to ascertain

      😅

    3. increased activity has brought with it a degree of hype and misinformation, which can undermine progress and public confidence in the research

      I think any field that has large implications in people's lives - such as food and health are susceptible to misinformation and hype. And it makes is especially complex to debunk when the actual science is too complex to communicate to general public while the field is still in progress

    1. Proteinase K to the reaction to a final concentration of 50–100μg/ml. Incubate at 37–56°C for at least 1 hour. R

      Is 1 hour necessary? The Maxwell protocol DNA Purification from Microorganisms in Water Samples mentions 70C for 10 min with ProK and RNAseA..?

    1. Differences in growth rate, e.g. induced by sub-lethal antibiotic concentrations or temperature, can also significantly bias conjugation rate estimates.

      Do growth conditions bias conjugation rate estimates or do they actually change conjugation rates? - I think it's the latter

    1. The default (50nM) works well with the standard protocol used at the Whitehead/MIT Center for Genome Research

      500 nM each oligo in reaction

    1. a rerun of those identical amplicons (from the same reaction tubes) on a different date yielded “normal” results

      This is scary. How do we trust anything sequencing?

    1. A normal blood sodium level is between 135 and 145 milliequivalents per liter (mEq/L).

      What the fuck are these units? Why not just saw mM?

    1. adjust the sample richness of each ecosystem by adding to it an estimate of the number of unobserved species

      Is this a straightforward thing to do? - Are there any user choices reg distribution etc. that need to be made?

    2. Adjusting for sample size when comparing different groups of observations without discarding data is widely prevalent in the sciences, and discarding data to adjust for unequal sample sizes is the exception

      Ok, I'm sold. No rarefying anymore :P

    3. there is substantial negative bias in the estimates

      rarefaction creates a negative bias?

    4. since estimates for alpha diversity metrics are heavily biased when taxa are unobserved, comparing alpha diversity using either raw or rarefied data should not be undertaken

      Can we say that observing or not of rare taxa depends on the depth of sequencing? (+ other stuff such as lysis method, sample processing etc.)

    5. We currently do not account for measurement error in microbial diversity studies.

      Good point. What do I do with biological replicates?

    6. unique property of microbiome experiments and alpha diversity analysis is that samples do not faithfully represent the entire microbial community under study

      Saying that samples are not representative?

    7. The samples are not of particular interest, except that they reflect the environment from which they were sampled

      Subsampling from a population

    1. Based on these results and well-established statistical theory, we advocate that investigators avoid rarefying altogether. We have provided microbiome-specific extensions to these tools in the R package, phyloseq.
    2. Well-established statistical theory is available that simultaneously accounts for library size differences and biological variability using an appropriate mixture model

      how do we really know what is the appropriate mixture model to use? - Considering that these are to be applied by biologists who are not too well trained in statistics?

      Is there any way to prove that any mixture model performs better than the default rarefaction etc.?

    1. since the most commonly  used models are underdispersed relative to the data, standard errors are almost always zero. The result is that ecologists see incredibly small p values, but replicating results is rare.

      Classical ecology models that work for 100-200 specimens don't work with microbial high throughput sequencing of thousands or millions of sequences, due to their less complexity and the underdispersed nature - Read more about the dispersion

    2. By the way this an example of an excellent lab website/research section with different pointers for different scientific fields and general public also!

    3. We care about producing high-quality software and making our papers reproducible.

      Wonderful :)

    1. the key aspect of a protocol’s ability to accurately reflect the original sample composition can only be assessed with the help of standardized controls like mock communities

      Might be relevant to the RNA memory vs FACS with mock community experimental design of the mock community

    1. sRNAs that repress transcription have been engineered to create orthogonal and composable regulators that can be used to construct RNA-only transcriptional networks
    1. techniques, such as transposition with Mu 11, Tn5 12 and Tn7 13, have been used to add fluorescent gene markers to plasmids

      Read these

  8. Aug 2023
    1. Alongside this, we used pKJK5::gfp, a plasmid lacking these additional payload genes

      review may have asked for this?

    1. none of the organisms identified are known to produce particularly small cells that could pass 0.2 µm filters

      Could broken membrane pieces from dead cells protect the DNA from DNAses?

    1. Nanopore sequencing is used to rapidly and inexpensively assess part composition and create a composition-to-barcode index

      Less coverage needed..

    1. For the terminus, we used primers that amplified part of the dcp gene: 5′-TTGAGCTGCGCCTCATCAAG-3′ and 5′-TCAACGTGCGAGCGATGAAT-3′

      TerC: qPCR copy number of e coli chromosome

    1. Capra Biosciences harnesses the superpowers of a biofilm-forming organism Marinobacter atlanticus to make fat-soluble products like retinol which is used in anti-aging face creams, and high-end industrial lubricants for electric car engines
    2. Some startups, like MicroByre, have been working to domesticate those reluctant, recalcitrant, and rebellious microbes by adapting them for use in the lab and creating genetic tools to manipulate them
    1. Filter and trim

      Also recommended trimleft = 10

      We also choose to trim the first 10 nucleotides of each read based on empirical observations across many Illumina datasets that these base positions are particularly likely to contain pathological errors. Source

      Also note

      Trimming and filtering is performed on paired reads jointly, i.e. both reads must pass the filter for the pair to pass.

      Callahan, Ben J., et al. "Bioconductor workflow for microbiome data analysis: from raw reads to community analyses." F1000Research 5 (2016).

  9. Jul 2023
    1. binding of the Mob protein to the 52-bp sequence could thus allow the formation of a protein-DNA complex with a double function: relaxosome formation andmob gene regulation.

      Mob binds to it's own promoter and represses (negative regulation)

      This is why Mob is preferred in cis for this dual action? - to not make too much mob?

    1. Student's t-Test

      is used to test the (null) hypothesis that two populations have equal means. Source Welch t-test: Wikipedia

    1. approaches, which typically encapsulate bacteria in hydrogels, have produced deployable optical sensors for explosives14, heavy metals15 and chemical inducers16,17
    1. Translocation to a cell’s surface utilizes a signal peptide (for inner membrane translocation) and AIDAc as an outer membrane autotransporter pore
    1. AHL communication modules with functional devices built from the biological components of the las18, tra18, rpa18, rhl19, cin19 and esa20 quorum-sensing systems
    1. Receiver plasmids contained the synthetic PlasR promoter fused to a strong RBS (BBa_B0034) and GFP gene (BBa_E0040) from the IGEM registry. These plasmids additionally contained a pMB1 origin from pET28a and either a chloramphenicol (CmR) or a kanamycin (KanR) selectable marker.

      pSH001 plasmid

  10. Jun 2023
    1. folA gene from the evolved and naive populations was amplified by PCR

      Did you not include the regulatory region?

    2. Evolution in regulatory regions rapidly compensates the cost of nonoptimal codon usage

      Did you observe this effect in the study?

    3. masked by mechanisms affecting the abundance and function of the protein products of the transferred genes downstream to transcription and translation steps, and, therefore, being unrelated to codon bias

      Same effect would also impact this study as well right?

    4. very high antibiotic concentrations used in this study (up to 30-fold the minimal inhibitory concentration [MIC]) potentially minimized the effect of sequence composition on fitness

      Is this by reducing the window of fit phenotypes?

    5. Computational studies that analyzed HGT events among bacterial genomes revealed that HGT frequency positively and strongly correlates with the similarity of tRNA pools between donors and acceptors
    6. 5′-end mRNA, the fitness contribution of mRNA folding stability dominates over that of codon optimality.

      Folding stability is universal between organisms!

    1. We constructed an Escherichia coli donor strain that can deliver a genetic payload into target recipients by broad-host-range bacterial conjugation

      How is this different from MFD-pir?