Lysis

About: Lysis is a research topic. Over the lifetime, 6072 publications have been published within this topic receiving 216978 citations.

A microfluidic bioreactor based on hydrogel-entrapped E. coli: cell viability, lysis, and intracellular enzyme reactions.

Abstract: Viable E. coli cells were entrapped in hydrogel micropatches photopolymerized within microfluidic systems. The microfluidic channels and the micropatches have sizes on the order of 100−500 μm. Small molecules, such as dyes and surfactants, present in the solution surrounding the hydrogel, are able to diffuse into the gel and encounter the cells, but the cells are sufficiently large to be retained. For example, sodium dodecyl sulfate is a lysis agent that is able to penetrate the hydrogel and disrupt the cellular membrane. Entrapment of viable cells within hydrogels, followed by lysis, could provide a convenient means for preparing biocatalysts without the need for enzyme extraction and purification. Hydrogel-immobilized cells are able to carry out chemical reactions within microfluidic channels. Specifically, a nonfluorescent dye, BCECF-AM, is able to penetrate both the hydrogel and the bacterial membrane and be converted into a fluorescent form (BCECF) by the interior cellular machinery. These results su...

Integrated microfluidic cell culture and lysis on a chip

Abstract: We present an integrated microfluidic cell culture and lysis platform for automated cell analysis that improves on systems which require multiple reagents and manual procedures Through the combination of previous technologies developed in our lab (namely, on-chip cell culture and electrochemical cell lysis) we have designed, fabricated, and characterized an integrated microfluidic platform capable of culturing HeLa, MCF-7, Jurkat, and CHO-K1 cells for up to five days and subsequently lysing the cells without the need to add lysing reagents On-demand lysis was accomplished by local hydroxide ion generation within microfluidic chambers, releasing both proteinacious (GFP) and genetic (Hoescht-stained DNA) material Sample proteins exposed to the electrochemical lysis conditions were immunodetectable (p53) and their enzymatic activity (HRP) was investigated

Release of transforming plasmid and chromosomal DNA from two cultured soil bacteria.

Abstract: The release of chromosomal and plasmid DNA from Acinetobacter calcoaceticus and Bacillus subtilis cultivated in minimal medium and broth over a period of 50 h was monitored and related to growth phase, autolysis, DNase production and natural competence. The released DNAs were biologically active in natural transformation. In addition, the circular integrity of a released B. subtilis shuttle vector (pHV14) was demonstrated by artificial transformation of Escherichia coli. In cultures of both strains high molecular weight DNA accumulated, particularly during the stationary and death phase (up to 30 micrograms ml-1). Generally, despite the presence in culture fluids of DNase activity (and of an intracellular enzyme, catalase, indicating some cell lysis) there was high transforming activity of chromosomal and plasmid DNA even 40 h after the cultures reached the stationary phase. In cultures of B. subtilis in minimal medium a presumably active release of intact plasmids and chromosomal DNA occurred during the competence phase. The release of biologically functional DNA during essentially all growth phases of a gram-positive and a gram-negative member of soil bacteria might facilitate horizontal gene transfer by transformation in natural habitats.

Rapid method for the purification of DNA from subgingival microorganisms.

Abstract: A method is described which facilitates the rapid purification of high molecular weight chromosomal DNA from gram positive and gram negative bacteria grown on solid media. A total of 32 reference strains and fresh isolates were examined in this study. The purification procedure involved lysis of cells with SDS in the presence of proteinase K, followed by removal of cellular polysaccharides and proteins with hexadecyltrimethyl ammonium bromide (CTAB) and phenol:chloroform:isoamyl alcohol. Preparations were incubated with RNase and, after removal of the enzyme, DNA was precipitated with ethanol. Several hundred micrograms of DNA could be prepared within 5 h from cells grown on 1-2 agar plates. None of the final preparations contained RNA; protein was detected in 12/32 preparations. The resultant DNA proved suitable for restriction enzyme digestion and biotin-labelling by a random primer technique. DNA probes constructed from these preparations were capable of detecting 100 pg of homologous target DNA fixed to nitrocellulose. Cross reactions between closely related species displayed weaker signal intensities than, and, thus, were easily distinguished from, true positive reactions between homologous species. DNA obtained by this procedure may also be suitable for DNA-DNA homology studies, recombinant DNA experiments and molecular fingerprinting.

Biosynthesis and disulfide cross-linking of outer membrane components during the growth cycle of Chlamydia trachomatis.

Abstract: The synthesis and accumulation of Chlamydia trachomatis outer membrane proteins within infected HeLa 229 host cells were monitored by assessing the uptake of [35S]cysteine into chlamydial proteins during the 48-h growth cycle of a lymphogranuloma venereum strain, L2/434/Bu. Synthesis of the major outer membrane protein, a protein that accounts for about 60% of the outer membrane protein mass of elementary bodies (EB), was first detected between 12 and 18 h after infection. The uptake of [35S]cysteine into the 60,000-molecular-weight doublet (60K doublet) and 12.5K cysteine-rich proteins was not observed until 30 h after infection, when the intracellularly dividing reticulate bodies were beginning to transform into infectious EBs. By using a more sensitive immunoblotting method in conjunction with monoclonal antibodies specific for the 60K doublet proteins, synthesis of these proteins was detected even earlier, by 18 h after infection. These data suggest that the time and extent of synthesis of these outer membrane proteins are regulated by processes that coincide in time with the transformation of reticulate bodies into EBs. Additional studies were performed to determine the extent of disulfide cross-linking of outer membrane proteins during the growth cycle. Both the major outer membrane protein and the 12.5K protein became progressively cross-linked to about 60% during the last 24 h of the growth cycle, whereas the 60K doublet proteins were extensively cross-linked during most of the cycle. These data may indicate an intracellular cross-linking mechanism, possibly enzymatic, that exists in addition to an auto-oxidation mechanism that occurs upon host cell lysis and exposure to the extracellular environment.