adjustments must be made in the IGS to allow the formation of a stable P10 helix
How can you adjust the IGS when there are two constrains on it from both P1 and P10?
adjustments must be made in the IGS to allow the formation of a stable P10 helix
How can you adjust the IGS when there are two constrains on it from both P1 and P10?
best design delivers a 93-fold dynamic range of splicing with RENDR controlling fluorescent protein production in response to an RNA input
wasn't the dynamic range much higher (10^4 fold?!) when we checked with qPCR?
Here is the information you may need in order to complete page 2 of the Form I-983:
If your employer is Rice University, here's the E-verify info -
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Quantification of native and barcoded 16S rRNA in E. coli expressing each cat-RNA using RT-qPCR.
explain what normalized RNA copies
means
We demonstrate here that bulk protein content partitions to wastewater solids. Using a combination of western blotting, ELISA, and mass spectrometry, we identify a robust repertoire of intact human antibodies, predominantly secreted IgA
Wastewater has a lot of junk that could bind to the sandwich ELISA non-specifically.
To validate this, I was wondering if there would be a good negative control antigen binder that you could look for - for example, some Ebola antibodies that you won't expect to be in this wastewater?
used model strains Escherichia coli (E. coli) K12 MG1655 as donors and recipients along with the IncPα model conjugative plasmid RP4
Does this effect still matter if using an auxotrophic donor such as MFD-pir?
However, it is unknown whether PFASs affect the HGT of bacterial antibiotic resistance.
This is a very poor rationale for why it should be studied. There are many things that are unknown, that does not mean they are worth knowing
At all stop signs, cyclists must stop and yield the right-of-way to other vehicles and pedestrians already at the intersection. RUPD will ticket cyclists for right-of-way violations at intersections.
So if there is no other entities, the bikes don't need to stop?
Could this be regarded as a Yield
sign instead?
Registration helps RUPD to identify owners of lost, stolen or impounded bicycles and to disseminate safety information
Have there been any examples or recovering stolen bicycles by RUPD yet?
Bacteria isolated from humans and livestock are much more likely to have duplicated antibiotic resistance genes
Is this controlled for other factors? Such as more bacteria being isolated from these environments than others?
Recipient community cells were sorted from the same samples using the same conditions, including both colorless recipient and green fluorescent transconjugal cells
Would classifying the non-uptaking community as "recipients" rather than the initial starting community cause confusion?
Plasmid transfer was detected both in abundant and rare taxa of the initial recipient community
were there any OTUs that did not appear in the initial recipient community at detectable levels??
Venn diagram of the number of genera identified in recipient and the corresponding transconjugant pool in the non-pharmaceutical control
The transconjugant pool has genera that don't overlap with the recipient pool. How do you explain this?
Conjugation events were visualized by a confocal laser scanning microscope
Why didn't you quantify from the flow cytometry runs?
applied saliva from P- and NP-caterpillars to plant wounds and then assayed for a subset of the plant-defense genes including PIN2, TD2, and AspPI and the defense protein PPO
How did you chose this subset?
Reverse primer (10 µM)
primers: 0.5 uM final (same as NEB lunascript)
Useful discussion on the pros and cons!
development of a flow cytometry optimized GFP variant, gfpmut3 (Cormack et al., 1996), which is still extensively used for monitoring the fate of plasmids in natural environments.
what about the variant causes it to be optimized for flow cyt?
This corresponds to more sequences than sorted transconjugants for most samples (Supplementary Table 1), providing an adequate picture of the observed plasmid transfer range.
# of final reads / # of Tc sorted > 1
is that they mean
gate for only particles of bacterial size
How do you determine 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
ll label_() functions return a "labelling" function, i.e. a function that takes a vector x and returns a character vector of length(x) giving a label for each input value.
This function when called seems to return an expression rather than a character vector.
Test using this and compare to label_scientific
which works as intended
``` r
scales::label_log(digits = 1)(c(1, 10, 100))
scales::label_scientific(digits = 1)(c(1, 10, 100))
```
<sup>Created on 2024-07-31 with reprex v2.1.0</sup>
human cells could serve as an orthogonal system for studying PopZ condensation outside of the context of its Caulobacter binding clients
Wouldn't using a gamma-proteobacteria like E. coli be easier?
to improve the accuracy of taxonomic assignment at the species level for full-length 16S rRNA sequences, we manually curated the three databases and removed the sequences that did not have a species name
So these sequences won't be classified anymore?
for example, Vandeputte et al. (2017) measured total-community abundance using flow cytometry
how did you measure abundance properly without noise from particulates?
However, the abundance of one species may not influence the abundance of another; the area may contain both tigers and ladybugs, and the migration of several ladybugs into the area would not be expected to affect the number of tigers. The assumption of true independence can not hold in high-throughput sequencing (HTS) experiments because the sequencing instruments can deliver reads only up to the capacity of the instrument.
good example, compositional data
Its -best_hit_overhang parameter, H, controls when an HSP is considered short enough to be filtered due to presence of another HSP
Does this parameter help return a single/best match when blasting against a custom database?
echo "AATGTACTAT" | tr 'ATCGatcg' 'TAGCtagc' | rev
More thorough version is to account for all the IUPAC degenerate DNA codes like this
bash
echo sequence | tr '[ATUGCYRSWKMBDHVNatugcyrswkmbdhvn]' '[TAACGRYSWMKVHDBNtaacgryswmkvhdbn]' |rev
Assume your fragment of interest is mysequence and the adapter is ADAPTER. The reads may look like this:
5' adapter example -g
up to 90 days before your current OPT employment authorization expires, and within 60 days of the date your designated school official (DSO) enters the recommendation for OPT into your Student and Exchange Visitor Information System (SEVIS) record.
I guess it means dso enters the recommendation for STEM OPT
into the SEVIS record?
5GHz band
The latter adds speed but lacks range.
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
Where does this overhead come from?
Despite its utility, man is not always the answer. Sometimes grepping the help prompt for a term is all one needs.
Good idea!
"IAM & admin" -> "Service Accounts".
To navigate here: Credentials
(left menu) -> Service Accounts
-> Manage service accounts
In Linux, port numbers below 1024 are reserved for well-known services and can only be bound to by root. Although you can use a port within a 1-1024 range for the SSH service to avoid issues with port allocation in the future, it is recommended to choose a port above 1024.
why recommended above 1024?
translocation occurred from an IncF plasmid into a cryptic conjugative plasmid showing how cryptic conjugative plasmids are a significant concern because they can capture and disperse ARGs from the vast gene pool in the environments
Why only cryptic? Any conjugative plasmid can do the same?
The sensitivity of the assay is estimated to be 90% as 10% of the influenza A H5 subtype sequences in GISAID and NCBI have single nucleotide polymorphisms in the primer and probe regions.
This change might cover that additional 10%? - AGTGGKTAYGCTGCRGAC (note the Y in the middle is bolded there) instead of having a C in that position
Source: Mike Nute/Treangan lab @Rice
Btw in that boems paper they report their assay as 90% sensitive. I’m fairly sure that the C in that position is what is causing that 10% loss, so they may have chosen not to make that a Y for some very good reason because all the other ambiguous bases are based on much lower frequency variants, so I would suspect they tried a Y there somehow.
Do not exceed 5,000 copies of target/μl of the final ddPCR reaction mix
1e5 targets per 20 ul reaction well. Sounds a little high no for 20,000 droplets, I would estimate a total of 2,000 copies to keep the avg 0.1 copies per droplet?
Do not heat inactivate at greater than 65°C
heh, why? Older enzymes have 80C inactivation temps..
Many assays will perform well regardless of which NEBuffer is used or the amountof 1x digest reaction loaded into ddPCR
contradicts the above point about max 2 ul template / 22 ul rxn but ok..
Restriction Digestion
See notes in this section for restriction digestion tips
we compared the sensitivity of qPCR, HRM and dPCR in detecting the allele A from two pools of bulk beet DNA composed of 90 biennial + 10 annual plants (B1) and 99 biennial + 1 annual plant (B2), respectively
Read about probe design - One probe per allele?
Samples must be received by 3 pm ET Wednesday
Which means, samples should be submitted by 3 pm Tuesday to Genewiz dropbox. - Remember that it takes 30 mins to fill the Amplicon EZ form and 15 mins to do the qubit and dilutions too
There could be some delays in shipping, so it is best to have it picked up on Monday if timeline is urgent
low TE (<0.1 mM EDTA)
IDTE buffer from IDT should work
IDTE (10 mM Tris, 0.1 mM EDTA). Source: IDT
We recommend using a dsDNA quantification assay such as Qubit or PicoGreen. Note that the NanoDrop cannot distinguish between dsDNA and ssDNA (oligos and dNTPs) and may cause you to overestimate the amount of dsDNA in your samples.
Is nanodrop
ok to use then after size selection with mag beads?
HTML Options
This was not intuitive but these options go under this section shown below and this Posit community page was very helpful
format:
html: ..
You can use inline R code (see Section 3.1) anywhere in an Rmd document, including the YAML metadata section. This means some YAML metadata can be dynamically generated with inline R code, such as the document title
Does this apply to .qmd
documents?
Absolute quantitation of microbiota abundance in environmental samples
use of synthetic DNA spikes enables absolute quantification of microbiome members directly in environmental samples [14].
buffers with high salt
Dilute the restriction enzyme using the recommendeddiluent buffer
Did you mean reaction buffer? Because diluent is different from the reaction buffer. For HindIII this is diluent B
DNA fragmentation by restriction digestion prior to dropletgeneration enables optimal accuracy by separating tandemgene copies, reducing sample viscosity, and improving templateaccessibility for input samples >66 ng per well
NEB says
Digestion is recommended whenever DNA input is greater than 75 ng Source
NEB says that biorad recommends these enzymes: AluI, CviQI, HaeIII, HindIII-HF, MseI
More guidelines
- Assemble ddPCR reactions at room temperature, this allows the restriction enzyme to digest the gDNA during the reaction set-up period
- Prepare reaction mixes as you would for a standard ddPCR reaction. Add 0.5–1 μL of each restriction enzyme (5–20 units, depending on enzyme concentration) to the reaction mixture
- After set-up, simply continue droplet generation as normal
- Restriction enzyme will be inactivated during first PCR denaturation step
Diluent Buffers (A, B or C) are recommended for making dilutions of restriction endonucleases. When necessary, we recommend diluting enzymes just prior to use and suggest that the final concentration of diluted enzymes be at least 1,000 units/ml
How is the diluent better than the reaction buffer?
Digestion is recommended whenever DNA input is greater than 75 ng
Might be good to do on a cell lysate since there is unknown amount of DNA before direct ddPCR
Refer to this protocol mentioned in the BioRad page, annotation
The gun has been called the great equalizer, meaning that a small person witha gun is equal to a large person, but it is a great equalizer in another way,too. It insures that the people are the equal of their government wheneverthat government forgets that it is servant and not master of the governed
Is this questioning the monopoly of violence of the govt?
The sample can be periodically mixed by pipetting/vortexing/shaking to ensure thesbeadex particles remain in suspension. This mixing may increase the efficiency ofthe binding/washing
I noticed that the suspension is still brown enough without mixing for 5 mins
The composition of Buffer EB is: 10 mM Tris-Cl, pH 8.5
Size optimized Low settling rates for automation applications
avoids the need for agitation during binding/elution steps
cell pellet suspended in 20 μl of Lyse and Go PCR Reagent (Thermo Scientific)
Does this kind of lysis bias the community to easy lysers? - I'm currently just heating 95C, 10 m for lysis and worry about the same. Will switch to not pelleting cells, adding a lysis buffer and proceeding into a mag bead based DNARNA extraction kit (Maxwell)
The more recent method used by Klumper paper, 2022 uses a proper extraction kit but also acquires more cells -
A minimum of 50,000 cells were acquired in all sorting runs.The sorted transconjugant and recipient cells were lysed and DNA extractions were performed using the DNeasy Powersoil Pro Kit. Source: Wang, Yue, et al. "Non-antibiotic pharmaceuticals promote conjugative plasmid transfer at a community-wide level." Microbiome 10.1 (2022): 124.
Thermo discontinued lyse n go. alternatives? - check Microlysis from gelcompany
centrifuged at 10 000 g for 30 min to collect the cell pellets
That's quite long..
Keep sorted transconjugants in 30% glycerol at 4C
Why glycerol?
3 mL pyrophosphate buffer
Pyrophosphate buffer: 50 mM Na 4O 7 P2 , 0.05% Tween 80; adjust pH to 8.0. Sterilize by autoclaving.
making our analyses available through a Shiny app and R package
Could be a good activity to cover in a statistics class!
Mix thoroughly and incubate for 5 minutes with periodic shaking
How essential is this periodic shaking? OmegaBiotek's similar protocol doesn't have this
- Pipet up and down 5-10 times or vortex for 30 seconds.
- Let sit at room temperature for 5 minutes. source: Manual for Mag-Bind® TotalPure NGS
Google Photos ended its unlimited free storage policy for photos and videos as of June 1
June 1, 2021
Any Wanna Get Away or Wanna Get Away Plus funds will be forfeited
only
If you don’t cancel your reservation at least 10 minutes before the flight’s original scheduled departure time
No change fees. We never charge a fee to change your flight
for all southwest fares, including the cheapest Wanna get away
replaced the Taq polymerase used for primary amplification of the unique molecular identifier (UMI) tagged templates with a proofreading polymerase (Supplementary Fig. 16). This increased the percentage of error-free reads nearly four-fold, which greatly improves our ability to resolve full-length 16S rRNA ASVs (FL-ASVs) from low abundant taxa because at least two identical sequences are required to resolve an FL-ASV
Phusion polymerase. Tested on ZymoBIOMICS Microbial Community DNA Standard
1391r primer48 as it has better coverage of the known bacterial diversity
Figure out how much this is better by
Dueholm, M. S. et al. Generation of comprehensive ecosystem-specific reference databases with species-level resolution by high-throughput full-length 16S rRNA gene sequencing and automated taxonomy assignment (AutoTax). mBio 11, e01557–20 (2020).
why ecosystem specific databases vs general ones?
ASVs are not ideal as references for linking microbial identity with the physiology and ecology of key community members
As opposed to a taxonomic assignment
India
Indian citizens are required to have a visa to enter Canada.
if your treatments are ordered, don't compare each mean with each other mean (multiple comparisons), instead do one test for trend to ask if the outcome is linearly related with treatment number
How do you do hypothesis testing for trends for an ordered categorical variable?
Could you convert x
to numbers (1,2,3) and run a linear regression y ~ x? or even categorical ordered variables can be linearly regressed?
e are typically not terribly concerned with Type 1 error because werarely believe that it is possible for the null hypothesis to be strictly true.
What is this assumption based on?
NNK
Why NNK
as opposed to NNN
?
Supports up to 8GB maximum memory
only a single slot?
flea tips
new avenues for studying ARG transfer (Karimi et al., 2015; Li et al., 2018) (Figure 2C). Microfluidic chips equipped with delayed imaging, coupled with fluorescence technology, enable real-time observation of community changes and tracking of the transfer dynamics of ARGs
Scott Banta “Engineering of Acidithiobacillus ferrooxidans forBioelectrocatalysis Applications”
Great talk! held my interest for quite long Isobutyric acid made instead of intended isobutanol,..
Akihiro Okamoto “Decoding Extracellular Electron Transfer: A NewGlimpse into In-VivoEnzyme Dynamics
Very interesting idea : sl 13 - Liposome enters cells and grabs some outer membrane stuff including cytochromes that can do EET - Electron source: by X-ray striking an internal TiO2 nanoparticle! -> EET -> generate ROS -> cure a tumour!?
Biki Kundu
part notes on the web version - https://naismet2024.rice.edu/program/
10:20AM Biki Kundu
Excellent story. Worth emulating, Ask for a video recording!
lmer(heartrate ~ depth + duration + (1|bird), data = penguins)
Use this stats.stackexchange thread to understand the notation of the hierarchial / mixed effect mode
Use the lsb_release -a command to display the Ubuntu version.
quarto publish gh-pages document.qmd
Explore tokens
to minimize the number of ssh passphrase asked by this command!
A bracketed sequence of inlines, as one would use to begin a link, will be treated as a Span with attributes if it is followed immediately by attributes: [This is *some text*]{.class key="val"}
8.4.2 infer package workflow
A nice and easy way to report results of an ANOVA in R is with the report() function from the {report} package:
out-width Width of the plot in the output document, which can be different from its physical fig-width, i.e., plots can be scaled in the output document.
fig-width reference
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
I
I is ionosine? that matches to all nucleotides?
This preparation includes fragments from the RNA-dependent RNA polymerase and VP1 (ORF1-ORF2 junction) regions.
of which strain of Norovirus?
(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
git log -SFoo -- path_containing_change
Excellent command to search in git history of a specific file!
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.
This item type can also be adapted to fit many types of unusual items.
report
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?
Ensuring that each amplicon is only copied by its “correct” SuperSelective primer
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?
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?
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).
Underlined letters represent LNA bases.
I don't see any underlined bases here 😠
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?
SNP PI3Kca_E545K
How many nucleotides is this?
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
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?
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?
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?
10 ng Human PBMC gDNA from the same healthy donor was used per experiment, corresponding to 2,790 haploid copies
How was this determined?
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?
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?
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
With the file(s) selected, press Shift-Z
Linking to a file within multiple folders.
Sharing permissions are probably not carried over? check this
or changes between two files on disk
I assume they have to be in the same repo?
This approach exploits the equivalence between confidence intervals and hypothesis tests, detailed in Section 12.2.
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 = )
The data on the 463 courses at UT Austin can be found in the evals data frame included in the moderndive package
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
Incremental reveal Not limited to just bullet points... Use `. . .` syntax to indicate a break at an arbitrary location . . . ```{r} head(mtcars) ```
quarto incremental reveal
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
10,000 units
100,00 U/ 200 U/ul = 50 ul => 50 reactions using 200 units = 1 ul/20 ul rxn.
Benefit from increased resistance to inhibitors and chemicals with our optimized buffer system
Any other educational assistance that is excludable from gross income (tax free),
Can this be considered a definition of tax free?
Your filing status is married filing separately.
Cannot claim credit
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?
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?
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
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?
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
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.
Midterm
change to take home
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
git config --global alias.tree 'log --oneline --graph --decorate --all'
configuration to make a shortcut to show commits of git as a tree
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
. 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
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?
Decimal serial dilutions (100–1000 μl) of 101–106 or 107 were prepared from the 100 stock in 1.5 ml tubes with 4–5 repeated flushing and changing of tips
changing tips seems important?
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!?
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?
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?
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.
Place a space before and after () when used with if, for, or while.
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.
6c CAACGCGARGAACCTTACC 966–984
967F primer
though 8 V/cm (120 V) seems to work fine for routine use
Bennett lab has 15 cm gels
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
Summary of the experimental setup
Is there supplementary data in an intact mouse microbiome (without initial streptomycin treatment) for true in situ editing?
Dashed line: Background fluorescence of cells before transduction.
why not do a background subtraction?
the P2-STF chimera encoded on a plasmid (p938)
Why not the prophage genome?
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?
Efficient conjugative delivery to different strains and species will thus likely require different specialized systems
The same is true for phages as well
Full length Illumina tagged primers used in study:
27F (AGMGTTYGATYMTGGCTCAG) and ?R primer (GCTGCCTCCCGTAGGAGT) used
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.
What does a “model” mean?
R-course ; modelling intro
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
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
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.
515F GTGCCAGCMGCCGCGGTAA Universal Turner et al. 1999
Turner et al 1999 paper only worked with cyanobacteria right? How is this universal then?
PCR primers F-968-GC (59-CGC CCG GGG CGC GCC CCG GGCGGG GCG GGG GCA CGG GGG GAA CGC GAA GAA CCT TAC-39)
968f
968f968–984AACGCGAAGAACCTTACNübel et al., 1996
968F
In code, use comments to explain the “why” not the “what” or “how”.
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).
zygotically inducible
Interesting choice of terms!
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?
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
Conjugation events were visualized by stereomicroscopy and quantified by automated image analysis
Why didn't you use flow cytometry data for this?
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?
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?
(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)?
To be right, it must be possible to be wrong
something about over-fitting?
This model allows for the lessons to remain free from brand integrations and sponsor messages, without introducing paywalls.
Donation model
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.
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..
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
White space must be cropped from the image, and excess space between panel labels and the image must be eliminated.
how much is excess?
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
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)?
Any abuse of market power is likely to show up first on the supply side
Monopsony?
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)
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
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.
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)
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?
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
Note: option -u was replaced by -b in Mash v1.1.
and -m 2 in mash 2.0? source ; specific line
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
https://gembox.cbcb.umd.edu/mash/refseq.genomes.k21s1000.msh
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]
sample size must be between 3 and 5,000 in order to use the shapiro.test() function.
Does a sample size 3 really work?
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
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
30 - 60 sec/kb
Extension time
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
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.
Why is statistics difficult!? - Good example to separate immediately measured tangible variables and imaginary/statistical properties such as probability
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?
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.
mRNAs triphosphorylated at their 5′ ends.
Good illustration of the 5' triphosphate in Wikipedia - 5' cap structure ; image
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
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)
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"