The fundamental problem was that in the presence of the RDF, the recombinase action becomes bidirectional, since either the recombinase on its own or the recombinase-excisionase complex is able to flip DNA, each in a different direction

Although there aredozens of serine integrases that have been described in the literature, there are onlyseven published RDFs for serine integrases (C31 gp3 [22],BT1 gp3 [25], Bxb1 gp47[23], TP901 ORF7/Xis [24],Anabaena/NostocXisI[28], SPBc SprB [27], andRv1 Rv1584c/Xis [26])

piggyBac transposon has a broad host spectrum from yeast to mammals, this mobile element has been widely used for a variety of applications in a diverse range of organisms

homologs of the Tn7 transposition proteins, in highly diverged bacteria adapted to a remarkable number of different environments

incomplete (∼80%) conversion of an attP × attB plasmid substrate to recombinant products in vitro

revealed various types of damage, including mutations at the centres of the att sites (rendering them refractory to further reactions) and deletions at the sites which may extend into flanking DNA

However, serine integrases do show low levels of off-target activity at ‘pseudosite’ sequences that are similar to the natural att sites

uses of serine integrases for synthetic biology applications; for assembly of long arrays of genes or other DNA fragments, creation of genetic switches, circuits and logic systems, and for permanent genomic recording of cellular events

unique selling point of the latter systems is their one-way directional nature

attP and attB sites for serine integrases are both short (attP ∼50 bp; attB ∼40 bp); each site is bound by a dimer of integrase, and no host-encoded proteins are required

for specific stimulation of attL × attR recombination; often called Xis (‘excisionase’) for λ integrase and its tyrosine integrase relatives, or RDF (Recombination Directionality Factor) for serine integrases

DNA fragment assembly using site-specific recombination by ϕC31 integrase

Clearly, premature removal of the cI cassette in the absence of an inducer can limit environmental applications of this type of biosensor

availability of more nutrients in the rhizosphere than in bulk soil, and thus the higher metabolic activity of cells in the rhizosphere than in bulk soil, probably explains why the A506 (pTolLHB) biosensor cells responded to toluene on roots but not in the bulk soil.

inability of stationary‐phase biosensor cells to respond at all

microbial bioreporters (Table 2) report their perception of, or response to, their environments

stimulates spacer acquisition from double-strand breaks. This activity is limited by chi sites

sequence motifs that slow RecBCD activity

chronic infection whereby the viral genome resides within the cell

whole cells, treated by heating at 95°C for 10 minutes prior to storage at -20°C, were used as a template source

pUC origin carries a point mutation in the sense RNA that stabilizes the priming complex, resulting in higher copy number

improve DNA transformation

Surprisingly, quorum sensing promotes V. cholerae virulence factor expression and biofilm formation at low cell density and represses these traits at high cell density

Quorum sensing is essential for chronic P. aeruginosa respiratory infection because it controls adhesion, biofilm formation, and virulence factor expression

ampicillin

kanamycin

We conclude that these antibiotic exposures did not produce ROS

This is a direct result of the evolution of these antibiotics in microorganisms competing for resources, in which the production of antibiotics that target fast growing bacterial cells

Here we combined RNA-seq with an in vivo assay to identify the major transcriptional changes that occur in Escherichia coli when inducible synthetic constructs are expressed.

introduction of Cu to agricultural soil selects for Cu resistance, but also indirectly selects for antibiotic resistance in the Cu‐resistant bacteria

response to antimicrobial agents can greatly vary, depending on the location of a particular cell within a biofilm community

For example, bacteria could be engineered to seek out hazardous chemicals or heavy metals in the environment, perform cleanup and return to their origin to report on the number of hazardous sites encountered via analysis by microfluidic devices

operating in the frequency domain can offer considerable advantages over steady-state designs in terms of information gathering and transmission

less sensitive to environmental noise and exposure time than systems based on steady-state signals

QS represses biofilm formation in both S. aureus and V. cholerae

Pseudomonas aeruginosa is motile upstream (a QS-off phenotype), but forms biofilms downstream (a QS-on phenotype) in flowing networks

one to two AHL molecules per cell are sufficient to trigger autoinduction, suggesting that at very low AHL concentrations QS is intrinsically stochastic

attB and attP sites are typically ≤ 50 bp with different inverted repeat sequences flanking 2 to 12 bp of complementary sequence, which includes the recombination crossover point

240 μmol

absence of nitrogen was found to increase HGT events three orders of magnitude

in most cases, the molecular mechanisms that underlie heterogeneity are not defined

the Tn5 transposon has been shown to be functional in a wide range of Gram-negative bacteria and to randomly insert into their genomes with high efficiency

reporter response is highly dependent on culture age and cell population density

using the arabinose-inducible PBAD promoter because it has a tight OFF state while maintaining a large dynamic range

bistable autoregulatory transcriptional feedback

HiFi for sure.

we recommend using 45 nM of each oligonucleotide

using recombinases can be challenging because their reactions are slow (requiring 2–6 h) and often generate mixed populations when targeting a multicopy plasmid

added 5 μl of diluted oligo DNA (180 nM each)

following

the direct qPCR master mix, were added for direct qPCR.

we performed direct qPCR amplification

SYTO9

we had initially added a higher concentration of the aforementioned components

induced

qPCR components comprised

bovine serum albumin (BSA; Sigma, St. Louis, MO, USA), to adsorb PCR inhibitors

10–30 second annealing step at 3°C above the Tm of the lower Tm primer.

Lysate standard sample qPCR

Zandy, Zotpad, Scanner for Zotero, Bibup and Zotfile.

tudents must be registered for a minimum of 6 credit hours

exopolysaccharide > flagella >N-acyl-homoserine lactones (AHLs) signaling molecules > extracellular protein > swarming motility

using a commercially-available buffer that also requires a 15-minute incubation period prior to direct PCR

organisms require physical lysis mainly based on heat,(31) pressure,(32) sonication with sound waves,(33) and bead milling.(34)

The fluorescence signal atCT37±7 corre-sponds to the no-template-control andrepresents bacterial DNA contamination inthe commercially supplied reagents.

insulation of minimal promoters was demonstrated

each with 80% power (chance) of achieving P < 0.05

real-time information

Several aspects influence PCR yield and specificity: reagents concentration, primer and amplicon length, template and primer secondary structure, or G+C content

suggesting recent horizontal acquisitions

travelling

negative controls containing AF1000 cells devoid of plasmid yielded Ct numbers higher than 26 and were automatically censored by the LightCyclerTM software.

Fig. 4

a shrinking number of people have personal memories

peer-reviewed research

the difference in how scientists and the general public perceive risk

a loss of personal freedom

observed reduction was likely an underestimate due to the bimodal circuit activation in each population

observation of hysteresis

There are some techniques

Partial charges and discharges that combine to 100% are counted as a single full cycle

research says

low rates of indels (typically no more than 0.1%

push the technical limits of this information storage system

But the bully-pulpit talk didn’t do much

containing ROX reference dye