RP4 plasmid (also known as RK2, RP1, and the Birmingham plasmid) stands not only as a model of bacterial conjugation studied over the past 40 years, but also as one of the most conspicuous, broad-host range conjugative plasmids described in the literature. It mediates mating and plasmid transfer between a wide variety of Gram– donors/recipients (8) and is also capable of efficiently conjugating with Gram+, (9) yeast (10,11) and mammalian cells. (12)

Optimization parameters included: (i) molten agar media temperature, (ii) molten agar media agar concentration, and (iii) volumes of E. coli and S. cerevisiae cell suspensions harvested at various optical densities (ODs)

Some conjugation systems, such as IncF, IncH, and IncI plasmids, transfer DNA efficiently in liquid media, while others, including the IncN, IncM, IncP, and IncW plasmids, achieve higher DNA transfer frequencies on solid media [28]. It is suspected that the ability to transfer DNA in different environmental conditions is related to variation in pilus formation, structure, and stability of cells during the conjugation process.

such a protocol may permit more accurate enumeration of transconjugants by avoiding the use of a spreader while plating cells on selective media

may enhance the ability to transfer plasmids and chromosomes greater than 100 kbp

we developed a procedure for conjugation within solid media. Such a protocol may expand conjugation as a tool for DNA transfer to species that require semi-solid or solid media for growth

A. thaliana (hereafter referred to as Arabidopsis)

Over the last 25 years, shotgun metagenomic sequencing1 and associated computational methods have developed as robust, efficient ways to study the taxonomic composition

MetaPhlAn 4 relies on ~5.1M unique clade-specific marker genes identified from ~1M microbial genomes (~236,600 references and 771,500 metagenomic assembled genomes) spanning 26,970 species-level genome bins

Template Buffer should be at 1X inthe final reaction

not compatible with real-time PCR due to the presence of unnatural nucleotides in their sequence

removal of sequences containing nonsense mutations was found to be a valuable filter in addition to flowgram-based denoising

1.8 uL diluted ROX per 20 uL

We found a distinctive wheel-shaped arrangement of the cells

Studying gene–gene correlations on a local scale, we observed the expected high correlations between the expression profiles of genes residing in the same operon

Principal component analysis (PCA) at the gene level

Two-dimensional projection by uniform manifold approximation and projection (UMAP) of LB-grown E. coli

Degenerate consensual pairs of rpoB primers called Univ_rpoB_F_deg (forward primer) and Univ_rpoB_R_deg (reverse primer) were manually designed from clustalW alignments

we constructed a reference database including ~ 45000 sequences; this database is available from the FROGS website (http://frogs.toulouse.inra.fr/).

In many cases, such variations reflect differences in the essentiality of genes. Genes with higher connectivity are often more essential for the reproductive success of a cell or organism

poses challenges for many classical methods, such as parametric statistical tests (for example, Student’s t-test and ANOVA) and measures of correlation, including Spearman’s rank correlation, often leading to completely unacceptable false discovery rates above 90%

We recommend that these methods replace OTU-based approaches for all applications, except when it is necessary to combine sequence data that were generated using different technologies (that is, Illumina sequencing and 454 pyrosequencing) or with different primer sets, when mapping to a common reference database of full-length sequences is often still needed

The number of k-mers in each hash bin is tabulated

If rearrangement events and horizontal transfers are rare for the 16S rRNA gene, as is widely believed to be the case, then its true gene tree is likely to be a good approximation to the true phylogenetic tree based on vertically inherited traits, assuming that the latter tree can be meaningfully defined

If an environmental sequence is annotated as belonging to a taxon which is defined by traits, then this is a prediction which can always be checked in principle

highly multiplexed ribosomal (r)RNA-FISH

The diversity of bacteria can also be accessed by using COI, rpoB, cpn60 (encodes for chaperonin protein), tuf (elongation factor), RIF (Replication initiation factor), and gnd (Gluconate-6-phosphate dehydrogenase) gene as barcode

The origin of the rain is not clear. Rain often is attributed to delayed PCR onset [3] or partial PCR inhibition in individual droplets [4]. However, it could also be a consequence of damaged positive droplets with corresponding reduced fluorescence, or damaged negative droplets with increased background fluorescence, or a mixture of both [5].

we found that the clouds can become more compact and produce substantially less rain upon: (i) reducing the ramp rate to 1 °C/sec in every PCR step (Fig. 10a), (ii) increasing the annealing/extension time to 2 minutes (up from 1 minute; Fig. 10b), and (iii) increasing the number of cycles to 50 (Fig. 10c)28

Trans-splicing ribozymes can, in principle, target every uridine residue of a substrate RNA, but the target sites need to be accessible.

In this assay, the mRNA is incubated with trans-splicing ribozymes that carry a randomized internal guide sequence (IGS). This enables the ribozyme population to splice on every accessible site on the mRNA. The sites at which trans-splicing occurred were identified by RT-PCR, cloning, and sequencing

we performed RT-qPCR on the high-performing RENDR design, split site 15. We observed a 93-fold increase in the abundance of spliced mRNA in cells expressing both RENDR and RNA input compared to control cells lacking the RNA input

RENDR variants sensing different RNA inputs from RFP

best design delivers a 93-fold dynamic range of splicing with RENDR controlling fluorescent protein production in response to an RNA input

P1_loop_03 TAGTTACCTTT

P1_loop_03 TAGTTACCTTT

AAATAGCAATATTTACCTTTGGGTCA

adjustments must be made in the IGS to allow the formation of a stable P10 helix

Here is the information you may need in order to complete page 2 of the Form I-983:

Quantification of native and barcoded 16S rRNA in E. coli expressing each cat-RNA using RT-qPCR.

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

used model strains Escherichia coli (E. coli) K12 MG1655 as donors and recipients along with the IncPα model conjugative plasmid RP4

However, it is unknown whether PFASs affect the HGT of bacterial antibiotic resistance.

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.

Registration helps RUPD to identify owners of lost, stolen or impounded bicycles and to disseminate safety information

Bacteria isolated from humans and livestock are much more likely to have duplicated antibiotic resistance genes

Recipient community cells were sorted from the same samples using the same conditions, including both colorless recipient and green fluorescent transconjugal cells

Plasmid transfer was detected both in abundant and rare taxa of the initial recipient community

Venn diagram of the number of genera identified in recipient and the corresponding transconjugant pool in the non-pharmaceutical control

Conjugation events were visualized by a confocal laser scanning microscope

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

Reverse primer (10 µM)

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.

This corresponds to more sequences than sorted transconjugants for most samples (Supplementary Table 1), providing an adequate picture of the observed plasmid transfer range.

gate for only particles of bacterial size

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.

human cells could serve as an orthogonal system for studying PopZ condensation outside of the context of its Caulobacter binding clients

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

for example, Vandeputte et al. (2017) measured total-community abundance using flow cytometry

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.

Its -best_hit_overhang parameter, H, controls when an HSP is considered short enough to be filtered due to presence of another HSP

echo "AATGTACTAT" | tr 'ATCGatcg' 'TAGCtagc' | rev

Assume your fragment of interest is mysequence and the adapter is ADAPTER. The reads may look like this:

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.

5GHz band

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

Despite its utility, man is not always the answer. Sometimes grepping the help prompt for a term is all one needs.

"IAM & admin" -> "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.

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

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.

Do not exceed 5,000 copies of target/μl of the final ddPCR reaction mix

Do not heat inactivate at greater than 65°C

Many assays will perform well regardless of which NEBuffer is used or the amountof 1x digest reaction loaded into ddPCR

Restriction Digestion

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

Samples must be received by 3 pm ET Wednesday

low TE (<0.1 mM EDTA)

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.

HTML Options

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

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

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

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

Digestion is recommended whenever DNA input is greater than 75 ng

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

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

The composition of Buffer EB is: 10 mM Tris-Cl, pH 8.5

Size optimized Low settling rates for automation applications

cell pellet suspended in 20 μl of Lyse and Go PCR Reagent (Thermo Scientific)

centrifuged at 10 000 g for 30 min to collect the cell pellets

Keep sorted transconjugants in 30% glycerol at 4C

3 mL pyrophosphate buffer

making our analyses available through a Shiny app and R package

Mix thoroughly and incubate for 5 minutes with periodic shaking

Google Photos ended its unlimited free storage policy for photos and videos as of June 1

Any Wanna Get Away or Wanna Get Away Plus funds will be forfeited

No change fees. We never charge a fee to change your flight

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

1391r primer48 as it has better coverage of the known bacterial diversity

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).

ASVs are not ideal as references for linking microbial identity with the physiology and ecology of key community members

India

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

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.

NNK

Supports up to 8GB maximum memory

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”

Akihiro Okamoto “Decoding Extracellular Electron Transfer: A NewGlimpse into In-VivoEnzyme Dynamics

Biki Kundu

10:20AM Biki Kundu

lmer(heartrate ~ depth + duration + (1|bird), data = penguins)

Use the lsb_release -a command to display the Ubuntu version.

quarto publish gh-pages document.qmd

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.

There are a number of ways to include dynamic variables within documents rendered by Quarto

I

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

(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

git log -SFoo -- path_containing_change

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.

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

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

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)

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

Underlined letters represent LNA bases.

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

SNP PI3Kca_E545K

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.

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.

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)

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

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

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

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

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

With the file(s) selected, press Shift-Z

or changes between two files on disk

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

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

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

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