Capillary-composited microfluidic device for heat shock transformation of Escherichia coli

Sha, Jun; Wang, Yaolei; Wang, Jianchun; Ren, Li; Tu, Qin; Liu, Wenming; Wang, Xueqin; Liu, Ajing; Wang, Lei; Wang, Jinyi

doi:10.1016/j.jbiosc.2011.06.004

Cited by 6 publications

(5 citation statements)

References 22 publications

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“…To generate the mixed hydrogel beads in the microuidic device, 50% (v/v) matrigel in DMEM either with or without HeLa cells, 2 wt% sodium alginate in DMEM, and mineral oil were injected into the microdevice through the curved, side, and oil channels, respectively. Similar to previous studies, 44,45 droplets with tunable and uniform sizes were generated using this system (Fig. 2a-c).…”

Section: Mixed Hydrogel Bead Generation and Cell Encapsulationsupporting

confidence: 57%

Mixed hydrogel bead-based tumor spheroid formation and anticancer drug testing

Wang

2014

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Three-dimensional multicellular tumor spheroids have become critical for anticancer study since they may provide a better model than conventional monolayer cultures of cancer cells. Various methods for tumor spheroid formation have been explored. However, only one kind of hydrogel was used in these methods, which has an influence on the size and morphology of the obtained tumor spheroids. Herein, we present a microfluidic droplet-based method for the formation of multicellular tumor spheroids using alginate and matrigel mixed hydrogel beads. By on-chip changing the flow rate of the two hydrogel solutions, mixed hydrogel beads with different volume ratios between alginate and matrigel are obtained. Meanwhile, human cervical carcinoma (HeLa) cells are encapsulated in the mixed hydrogel beads. Acridine orange and propidium iodide double-staining assay shows that the viability of cells encapsulated in the mixed hydrogel beads was more than 90%. After 4 day culture, the multicellular tumor spheroids were successfully formed with spherical shape and uniform size distribution compared with spheroids formed in pure alginate beads. Cytoskeletal analysis by TRITC-phalloidin staining show that HeLa cells in the mixed hydrogel beads closely link to each other. The dose-dependent response assay of HeLa cell spheroids to vincristine show that multicellular spheroids have more powerful resistance to vincristine compared to conventional monolayer culture cells. Taken together, this novel technology may be of importance to facilitate in vitro culture of tumor spheroids for their ever-increasing utilization in modern cell-based medicine.

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Section: Mixed Hydrogel Bead Generation and Cell Encapsulationsupporting

confidence: 57%

Mixed hydrogel bead-based tumor spheroid formation and anticancer drug testing

Wang

2014

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“…The transformation of bacteria in emulsions has been reported (71,72). Large-scale emulsion-generation techniques to produce 10 8 –10 9 droplets are also known (73).…”

Section: Discussionmentioning

confidence: 99%

Prospective identification of parasitic sequences in phage display screens

Matochko

Tang

et al. 2013

Nucleic Acids Research

119

132

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Phage display empowered the development of proteins with new function and ligands for clinically relevant targets. In this report, we use next-generation sequencing to analyze phage-displayed libraries and uncover a strong bias induced by amplification preferences of phage in bacteria. This bias favors fast-growing sequences that collectively constitute <0.01% of the available diversity. Specifically, a library of 109 random 7-mer peptides (Ph.D.-7) includes a few thousand sequences that grow quickly (the ‘parasites’), which are the sequences that are typically identified in phage display screens published to date. A similar collapse was observed in other libraries. Using Illumina and Ion Torrent sequencing and multiple biological replicates of amplification of Ph.D.-7 library, we identified a focused population of 770 ‘parasites’. In all, 197 sequences from this population have been identified in literature reports that used Ph.D.-7 library. Many of these enriched sequences have confirmed function (e.g. target binding capacity). The bias in the literature, thus, can be viewed as a selection with two different selection pressures: (i) target-binding selection, and (ii) amplification-induced selection. Enrichment of parasitic sequences could be minimized if amplification bias is removed. Here, we demonstrate that emulsion amplification in libraries of ∼106 diverse clones prevents the biased selection of parasitic clones.

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“…Two fragments were linked to the pMAD with corresponding double enzymes digestion. The pMAD-based plasmid constructs were transferred into the E. coli 116 receptor state to clone by the heat shock transformation method (Sha J et al 2011). To verify the correctness of the cloned DNA fragment, Plasmids were digested by enzymes and ampli ed by PCR.…”

Section: Strains Constructionmentioning

confidence: 99%

Isocitrate dehydrogenase of Bacillus cereus is involved in biofilm formation

Zhao

Liu

Huang

et al. 2021

Preprint

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Tricarboxylic acid cycle (TCA cycle) is a central carbon metabolism pathway in prokaryotes and eukaryotes, and involved in matter metabolism and energy production. Isocitrate dehydrogenase (IDH), which is a key enzyme in the TCA cycle, participates in the formation of biofilms in Staphylococcus aureus by regulating the redox state inside the cell. At present, it remains to be clarified whether IDH is involved in the formation of Bacillus cereus biofilms. In this study, we found a gene icdH annotated as encoding IDH in the B. cereus genome, and cloned and expressed the protein encoded by this gene. The enzyme activity assay showed that the protein had IDH activity dependent on NADP+, indicating that this gene encoded an IDH. The mutant ΔicdH was obtained by gene knockout. Phenotypic analysis showed that the biofilm yield and sporulation rate of the mutant ΔicdH decreased. To reveal the role of IDH in biofilm formation, extracellular pH and citric acid content were measured. The results showed that a B.cereus 0–9 strain that lacked IDH exhibited accumulation of citric acid and acidification of the extracellular matrix. Given that citric acid is a metal chelator, the accumulation of citric acid may lead to a lack of metal ions in cells, resulting in reduced cell viability and affecting biofilm formation. Consistent with this hypothesis, the addition of excess Fe3+ restored biofilm formation in the mutant. These results suggest that IDH in B.cereus may regulate biofilm formation by modulating intracellular redox homeostasis. In addition, we found that the icdH deletion of B. cereus 0–9 resulted in the destruction of the stage I of sporulation process, and thus resulted in a reduced sporulation rate, which was significantly different from sporulation in B. subtilis caused by interruption of the stage I sporulation process due to icdH loss.

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Capillary-composited microfluidic device for heat shock transformation of Escherichia coli

Cited by 6 publications

References 22 publications

Mixed hydrogel bead-based tumor spheroid formation and anticancer drug testing

Mixed hydrogel bead-based tumor spheroid formation and anticancer drug testing

Prospective identification of parasitic sequences in phage display screens

Isocitrate dehydrogenase of Bacillus cereus is involved in biofilm formation

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