Design of the LOCKR system.

different fouling patterns were observed for replicate MBRs seeded with the same inoculum and maintained under identical operating conditions, with similar levels of biomass, soluble microbial products, and extracellular polymeric substances

achieved only a 10% increase of permeate flux when backpulsing was used in the filtration of a biological wastewater containing a high mixed liquor suspended solids

Figure 5

Figure 2. TMP profiles during the operation of two one-stage MBRs in two phases.

C8-HSL was chosen as a representative AHL molecule because it is one of the dominant AHL molecules in MBRs for wastewater treatment

a critical issue in MBR that awaits a technological advancement to solve is membrane biofouling, which leads to high energy consumption and thus high operating costs

TMP-Jump

Plasmids (IncP/N/W)with 373short and rigid pili only transfer efficiently on solid surfaces, unlike those with longand flexible 374pili (IncF/H/T/J),capable of transferring equally well in liquidand on solid surfaces[35,36]

identify mechanistic drivers of microbial activity, infer meaningful interaction networks, and rationally engineer microbial communities.

Next, a microfluidic device is used to co-encapsulate these clusters with gel beads, each containing uniquely barcoded forward 16S rRNA amplification primers

current microbiome profiling approaches such as metagenomic sequencing require homogenization of input material, which means that underlying spatial information is lost

recording past events, to study localization of single-cell transcriptional events within the gut

These carboxylated AHLs facilitated the transition from a short cell to filamentous growth, with an altered carbon metabolic flux that favoured the conversion of acetate to methane and a reduced yield in cellular biomass

Deletion of genes thatproduceN-acyl-L-homoserine lactone signals resulted in an increase in denitrification activity, which wasrepressed by exogenous signal molecules.

The prokaryotic CRISPR-Cas system of defense against mobile elements seems to function via a bona fide Lamarckian mechanism

scientists are uncovering and debating an increasing array of other evolutionary processes at work in bacteria, some of which are suspiciously Lamarckian in character4–6

Instead, most particle-attached bacteria may be secondary consumers that recycle waste products from primary degraders.

co-occur out of cooperative relations or a similar niche preference

beneficial commensal

antibodies against siderophores in the intestine, which hindered proliferation of the gut pathogen Salmonella

At the micrometer-centimeter scale, electrochemical and optical probes have been productively used to profile gradients in pH, redox, and oxygen; however, these probes and their associated equipment are intrusive to the ecosystem and often expensive, limiting their application to only a few point measurements or limited time series, and their fragile nature makes them best suited for laboratory use

CustoMem in the UK uses synthetic biology to create a granular material that attracts and sticks to micropollutants such as pesticides, pharmaceuticals, and certain chemicals in wastewater

Now synthesized microbial biosensors are able to target specific toxins such as arsenic, cadmium, mercury, nitrogen, ammonium, nitrate, phosphorus and heavy metals, and respond in a variety of ways. They can be engineered to generate an electrochemical, thermal, acoustic or bioluminescent signal when encountering the designated pollutant.

remediating arsenic contamination of groundwater by stimulating natural bacteria to produce substances that arsenic sticks to

MicroBio Engineering Inc., a research and engineering consulting firm specializing in developing improved algal strains and cultivation processes for wastewater reclamation, animal feeds, higher value products and, yes, biofuels.

In Japan, scientists are developing synthetically altered soy plants and yeast that produce glycyrrhizen, a compound in licorice root that is 150 to 300 times sweeter than sugar

The modified freeze-squeeze method was more efficient in cleaning up small DNA fragments from agarose gels compared to commercial kits.

move away from identifying correlative relationships between characteristics of the microbiome and system-level processes, and towards identifying more causative and mechanistic relationships

endogenous AHL-based QS and QQ are existing and coexisting in biological wastewater treatments; the leading role for AHL-based QS or QQ phenomenon in biological wastewater treatments can be determined by bacterial AHL concentration.

, QQ has been successfully employed to reduce the pathogenicity of common plant pathogens (Zhang et al., 2007)

Rattanarat et al.73 created a three-dimensional μPAD (Fig. 1A) that combined colorimetric and electrochemical detection on separate layers for quantifying Ni, Cu, Fe, Pb, Cr(VI), and Cd

Immobilization efficiency was improved by manipulating the bacterial genetics (overproducing curli protein),

diagnosis of antibiotic interference in the major biosynthetic pathways of bacteria: the biosynthesis of DNA by the yorB promoter, of RNA by the yvgS promoter, of proteins by the yheI promoter, of the cell wall by the ypuA promoter, and of fatty acids by the fabHB promoter.

chief advantage of this approach is that the entire sensor, including all signal-processing and communication functions, can be produced as a single-chip, low-power, rugged, inexpensive device

Heat following

crude DNA extraction methods, such as boiling, are not recommended to obtain the lower detection limit for a microorganism

outstanding possibilities for studying phenomena such as competition, niche partitioning and predation in the microbial world, under more environmentally realistic conditions than can be achieved in pure culture studies.

the maintenance of the pluripotent state of animal stem cells requires hypoxic conditions, whereas higher oxygen tension promotes cell differentiation

broad‐scale functional parameters increased as biodiversity decreased, e.g. thymidine incorporation, growth on added nutrients, and the decomposition rate of plant residues. Other more specific parameters decreased as biodiversity decreased, e.g. nitrification, denitrification and methane oxidation.

But because they ignore so much of the data, they don’t work well with small sets of data

extremes help to tell you what’s possible, and could suggest a very different distribution in your data from what you expect

we observed that codon usage biases are better correlates of growth rates than any other trait, including rRNA copy number

capable of transferring resistance to non-resistant strains

The level of ARBs is extremely high in most of the river systems (up to 98% of the total detected bacteria)

relationships between characteristics of microbiomes and the system-level processes that they influence are often evaluated in the absence of a robust conceptual framework and reported without elucidating the underlying causal mechanisms. The reliance on correlative approaches limits the potential to expand the inference of a single relationship to additional systems and advance the field

Here, we show how three phenomena—Simpson’s paradox, context-dependence, and nonlinear averaging—can lead to erroneous conclusions about population parameters and species interactions when samples contain heterogeneous mixtures of populations or communities.

These issues can be overcome by measuring and accounting for spatial heterogeneity at very small scales

we emphasize how processes and interactions at one spatial or temporal scale contribute to emergent processes and properties at larger or longer scales. Most often the emergent processes we investigate are aquatic ecosystem services, including carbon burial, greenhouse gas emissions, nutrient removal, and recreational fisheries

some of the key questions of microbial community assembly, maintenance and function could be answered by intensively studying one (or a few) synthetic model community

Time-resolved analysis of individual cells revealed that past exposure to low levels of antibiotics increases tolerance to future exposure for the sessile but not for the motile cell

Now we can ask, ‘Have I seen iron at some threshold?’ ‘Have I seen some nitrate?’ And have every microbe write it down. As one thinks about long-term incubation, this is a really powerful tool, because even six months later we can pull out the DNA and see what happened.”

Growth history influences starvation-induced expression of uspA, grpE, and rpoS and subsequent cryotolerance in Escherichia coli O157:H7

in E. coli a form of ‘memory’ of past phosphate limitation leads to a faster response to successive periods of phosphate limitation, and that this faster response may be survival enhancing

This article discusses, and challenges, some of the often implicit assumptions made in community studies. It suggests greater focus on ecological questions, more critical analysis of accepted concepts and consideration of the fundamental mechanisms controlling microbial processes and interactions in situ

quantitative physiological characteristics such as maximum specific growth rate; saturation constant; optimum, minimum or maximum pH or temperature for growth; susceptibility to predation; and the speed of recovery after starvation.

qPCR

In minimal media, cell growth and division slow considerably, while protein overproduction from plasmids can continue

While these techniques offer good repression, they exhibit leakiness that precludes the gene of interest from being completely turned off

Or

Researchers could use the method to study poorly-understood aspects of the sulfur cycle or bacterial methane production

they upregulate biofilm formation and maturation (Sturbelle et al. 2015)

We overexpressed corresponding RT–Cas1 and Cas2 proteins from a plasmid that also contained their predicted CRISPR array

these technologies all require destruction of samples and prevent us from analyzing dynamic changes in molecular profiles, phenotypes, and behaviors of individual cells in a complex system

Live cell imaging is capable of analyzing spatiotemporal dynamics of molecules and cells with fluorescent proteins or probes, but only for a limited number of objects, which can be observed by microscopy

isolated Rsd inhibited transcription in vitro to various extents depending on the promoters used. We propose that Rsd is a stationary phase E. coli protein with regulatory activity of the sigma70 function.

promoter alignment of rpoS-dependent genes suggests a −10 consensus sequence for EσS [CTATA(A/C)T] basically identical to the Eσ70 consensus (TATAAT)

Thus, if the Birch effect carbon release is primarily from microbial materials, dry–wet cycling might reduce long-term carbon loss from soil. In contrast, if the Birch effect results from mobilizing otherwise stable old carbon pools, it would fuel microbial growth, possibly priming the loss of additional stable soil carbon.

It has long been recognized that rewetting a dry soil causes a pulse of CO2 (the Birch effect)

13C-acetic acid vapor

Dry soils can reduce microbial access to soluble resources, and drought stress can reduce the efficiency with which microorganisms use resources. But drying can also increase access to gaseous and volatile substrates

organisms that do not synthesize osmolytes are unlikely to grow in salty culture media

It is also unclear whether the flush of activity on rewetting is driven by microbial growth or by the physical/chemical processes that mobilize organic matter

explosion of findings that other bacterial species controlled genes for conjugation, exoenzyme production and antibiotic synthesis with luxI–luxR-like systems

a high-fidelity memory device might allow researchers to identify cell populations responsive to specific events and track their progression through the cellular response

suggested that low basal expression coupled with switch-like activation is required to maintain memory; growth rate was also found to significantly impact memory loop protein sustainability following cell division.

Several bacterial strains have been engineered to detect transient gene expression in the laboratory8,9,10,11 and in vivo for up to 12 d12,13

Transcripts with more polyadenylation are more likely to be translated compared to those without

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