E.coli DH5α cell response to a sudden change in microfluidic chemical environment
05 Nov 2015-Vol. 2015, pp 3213-3216
TL;DR: The studies revealed that when E.coli DH5α cells were exposed to 0.1 mM sorbitol, they showed faster chemotaxis towards the attractant and achieved steady state by 60 min, which shows that the bacterial cells respond to change in local chemical environment is within few minutes.
Abstract: Motile bacteria respond to changing chemical environment by moving towards or away from a particular location. Bacterial migration under chemical gradient is one of the most studied areas in biomedical field. In this work we looked into how bacterial cells respond to sudden change in the microfluidic chemical environment. E.coli DH5α cells were subjected to an attractant gradient (0.1 mM sorbitol - attractant to E.coli cells) and after 120 min the same cells were exposed to an inhibitor (0.1 mM NiSO 4 ) gradient in the same microfluidic device. Our studies revealed that when the E.coli DH5α cells were exposed to 0.1 mM sorbitol, they showed faster chemotaxis towards the attractant (0.1 mM sorbitol) and achieved steady state by 60 min. When we replaced 0.1 mM sorbitol with 0.1 mM NiSO 4 in the device we found that that the E.coli DH5α cells started responding to change in chemical environment within 10 min and achieved steady state at the end of 60 min. This shows that the bacterial cells respond to change in local chemical environment is within few minutes.
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TL;DR: Pilot experiments show that vectors only persist in indigenous populations when under selection pressure, disappearing when this carbon source is removed, which could prime indigenous bacteria for degrading pollutants while providing minimal ecosystem disturbance.
Abstract: Engineering bacteria to clean-up oil spills is rapidly advancing but faces regulatory hurdles and environmental concerns. Here, we develop a new technology to harness indigenous soil microbial communities for bioremediation by flooding local populations with catabolic genes for petroleum hydrocarbon degradation. Overexpressing three enzymes (almA, xylE, p450cam) in Escherichia coli led to degradation of 60–99% of target hydrocarbon substrates. Mating experiments, fluorescence microscopy and TEM revealed indigenous bacteria could obtain these vectors from E. coli through several mechanisms of horizontal gene transfer (HGT), including conjugation and cytoplasmic exchange through nanotubes. Inoculating petroleum-polluted sediments with E. coli carrying the vector pSF-OXB15-p450camfusion showed that the E. coli cells died after five days but a variety of bacteria received and carried the vector for over 60 days after inoculation. Within 60 days, the total petroleum hydrocarbon content of the polluted soil was reduced by 46%. Pilot experiments show that vectors only persist in indigenous populations when under selection pressure, disappearing when this carbon source is removed. This approach to remediation could prime indigenous bacteria for degrading pollutants while providing minimal ecosystem disturbance.
29 citations
TL;DR: The results show that the spherical AuNPs intervenes in the themotaxis of E. coli DH5α cells and inhibits the cell migration, which may be due to decreased F-type ATP synthase activity and collapse of membrane potential by Au NPs, which, in turn, leads to decreased ATP levels.
Abstract: Bacteria responds to changing chemical and thermal environment by moving towards or away from a particular location. In this report, we looked into thermal gradient generation and response of E. coli DH5α cells to thermal gradient in the presence and in the absence of spherical gold nanoparticles (size: 15 to 22 nm) in a static microfluidic environment using a polydimethylsiloxane (PDMS) made microfluidic device. A PDMS-agarose based microfluidic device for generating thermal gradient has been developed and the thermal gradient generation in the device has been validated with the numerical simulation. Our studies revealed that the presence of gold nanoparticles, AuNPs (0.649 μg/mL) has no effect on the thermal gradient generation. The E. coli DH5α cells have been treated with AuNPs of two different concentrations (0.649 μg/mL and 0.008 μg/mL). The thermotaxis behavior of cells in the presence of AuNPs has been studied and compared to the thermotaxis of E.coli DH5α cells in the absence of AuNPs. In case of thermotaxis, in the absence of the AuNPs, the E. coli DH5α cells showed better thermotaxis towards lower temperature range, whereas in the presence of AuNPs (0.649 μg/mL and 0.008 μg/mL) thermotaxis of the E. coli DH5α cells has been inhibited. The results show that the spherical AuNPs intervenes in the themotaxis of E. coli DH5α cells and inhibits the cell migration. The reason for the failure in thermotaxis response mechanism may be due to decreased F-type ATP synthase activity and collapse of membrane potential by AuNPs, which, in turn, leads to decreased ATP levels. This has been hypothesized since both thermotaxis and chemotaxis follows the same response mechanism for migration in which ATP plays critical role.
7 citations
TL;DR: In this article, the authors developed a new technology to harness indigenous soil microbial communities for bioremediation by flooding local populations with catabolic genes for petroleum hydrocarbon degradation, which could prime indigenous bacteria for degrading pollutants.
Abstract: Engineering bacteria to clean-up oil spills is rapidly advancing but faces regulatory hurdles and environmental concerns. Here, we develop a new technology to harness indigenous soil microbial communities for bioremediation by flooding local populations with catabolic genes for petroleum hydrocarbon degradation. Overexpressing three enzymes (almA, xylE, p450cam) in E.coli led to degradation rates of 60-99% of target hydrocarbon substrates. Mating experiments, fluorescence microscopy and TEM revealed indigenous bacteria could obtain these vectors from E.coli through conjugation. Inoculating petroleum-polluted sediments from a former refinery with engineered E.coli showed that the E.coli die after five days but a variety of bacteria received and carried the vector for over 120 days after inoculation. This approach could prime indigenous bacteria for degrading pollutants while providing minimal ecosystem disturbance.
2 citations
References
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TL;DR: Few microorganisms are as versatile as Escherichia coli; it can also be a highly versatile, and frequently deadly, pathogen.
Abstract: Few microorganisms are as versatile as Escherichia coli. An important member of the normal intestinal microflora of humans and other mammals, E. coli has also been widely exploited as a cloning host in recombinant DNA technology. But E. coli is more than just a laboratory workhorse or harmless intestinal inhabitant; it can also be a highly versatile, and frequently deadly, pathogen. Several different E. coli strains cause diverse intestinal and extraintestinal diseases by means of virulence factors that affect a wide range of cellular processes.
4,583 citations
TL;DR: Results are described which indicate that, when antibody-antigen complexes are incubated in fresh serum, a heat-stable substance (or substances) is produced which acts directly as a chemotactic stimulus on the polymorphs.
Abstract: An in vitro technique is described for assessing the chemotactic activity of soluble substances on motile cells. Antibody-antigen mixtures when incubated (37°C) in medium containing fresh (i.e. non-inactivated) normal rabbit serum exert a strong chemotactic effect on rabbit polymorphonuclear leucocytes. Results are described which indicate that, when antibody-antigen complexes are incubated (37°C) in fresh serum, a heat-stable (56°C) substance (or substances) is produced which acts directly as a chemotactic stimulus on the polymorphs. This heat-stable chemotactic substance is not produced when antibody-antigen complexes are incubated in serum which has been heated at 56°C for 30 minutes.
2,699 citations
TL;DR: Soft lithography offers the ability to control the molecular structure of surfaces and to pattern the complex molecules relevant to biology, to fabricate channel structures appropriate for microfluidics, and topattern and manipulate cells.
Abstract: ▪ Abstract Soft lithography, a set of techniques for microfabrication, is based on printing and molding using elastomeric stamps with the patterns of interest in bas-relief. As a technique for fabricating microstructures for biological applications, soft lithography overcomes many of the shortcomings of photolithography. In particular, soft lithography offers the ability to control the molecular structure of surfaces and to pattern the complex molecules relevant to biology, to fabricate channel structures appropriate for microfluidics, and to pattern and manipulate cells. For the relatively large feature sizes used in biology (≥50 μm), production of prototype patterns and structures is convenient, inexpensive, and rapid. Self-assembled monolayers of alkanethiolates on gold are particularly easy to pattern by soft lithography, and they provide exquisite control over surface biochemistry.
2,659 citations
TL;DR: Chemotaxis toward amino-acids results from the suppression of directional changes which occur spontaneously in isotropic solutions.
Abstract: Chemotaxis toward amino-acids results from the suppression of directional changes which occur spontaneously in isotropic solutions.
2,069 citations
TL;DR: These results show that E. coli is chemotactic toward oxygen and energy sources such as galactose, glucose, aspartic acid, threonine, or serine and that chemotaxis allows bacteria to find that environment which provides them with the greatest supply of energy.
Abstract: Motile Escherichia coli placed at one end of a capillary tube containing an energy source and oxygen migrate out into the tube in one or two bands, which are clearly visible to the naked eye and can also be demonstrated by photography, microscopy, and densitometry and by assaying for bacteria throughout the tube. The formation of two bands is not due to heterogeneity among the bacteria, since the bacteria in each band, when reused, will form two more bands. If an anaerobically utilizable energy source such as galactose is present in excess over the oxygen, the first band consumes all the oxygen and a part of the sugar and the second band uses the residual sugar anaerobically. On the other hand, if oxygen is present in excess over the sugar, the first band oxidizes all the sugar and leaves behind unused oxygen, and the second band uses up the residual oxygen to oxidize an endogenous energy source. The essence of the matter is that the bacteria create a gradient of oxygen or of an energy source, and then they move preferentially in the direction of the higher concentration of the chemical. As a consequence, bands of bacteria (or rings of bacteria in the case of agar plates) form and move out. These results show that E. coli is chemotactic toward oxygen and energy sources such as galactose, glucose, aspartic acid, threonine, or serine. The full repertoire of chemotactic responses by E. coli is no doubt greater than this, and a more complete list remains to be compiled. The studies reported here demonstrate that chemotaxis allows bacteria to find that environment which provides them with the greatest supply of energy. It is clearly an advantage for bacteria to be able to carry out chemotaxis, since by this means they can avoid unfavorable conditions and seek optimum surroundings. Finally, it is necessary to acknowledge the pioneering work of Englemann, Pfeffer, and the other late-19thcentury biologists who discovered chemotaxis in bacteria, and to point out that the studies reported here fully confirm the earlier reports of Beijerinck (4) and Sherris and his collaborators (5,6) on a band of bacteria chemotactic toward oxygen. By using a chemically defined medium instead of a complex broth, it has been possible to study this band more closely and to demonstrate in addition the occurrence of a second band of bacteria chemotactic toward an energy source. Beijerinck (4) did, in fact, sometimes observe a second band, but he did not offer an explanation for it.
1,116 citations