Heat-shock transformation of Escherichia coli in nanolitre droplets formed in a capillary-composited microfluidic device

Sha, Jun; Wang, Yaolei; Wang, Jian-Chun; Liu, Wenming; Tu, Qin; Liu, Ajing; Wang, Lei; Wang, Jinyi

doi:10.1039/c1ay05189j

Cited by 10 publications

(13 citation statements)

References 35 publications

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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%

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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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“…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%

“…We believe that it should be possible to use emulsion amplification to repair the collapse of diversity that occurs during the generation of libraries in bacteria. 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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“…46,47 All of these have been adapted to a microfluidic format, 48 and a smaller subset to droplet microfluidics. [37][38][39] The key parameters to determine success of this step in a microfluidic format are: yield, ease of implementation, ease of integration, and reproducibility. Heat shock is the easiest to implement in any microfluidic format, because the heating/cooling elements can be off-chip.…”

Section: Transformation/transfection Of Cellsmentioning

confidence: 99%

“…After construction of DNA libraries, screening these libraries for finding constructs with the desired activities remains a major scale-limiting bottleneck, both in terms of cost and time. Several strategies are available for delivering genes into cells including electroporation, 37,38 heat-shock, [39][40][41] microinjection, 42 cellular constraining, 43 sonoporation, 44 nanoparticles, 45 and viral transduction. 46,47 All of these have been adapted to a microfluidic format, 48 and a smaller subset to droplet microfluidics.…”

Section: Transformation/transfection Of Cellsmentioning

confidence: 99%

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Droplet microfluidics for synthetic biology

et al. 2017

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Emerging Droplet Microfluidics

2017

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Droplet microfluidics generates and manipulates discrete droplets through immiscible multiphase flows inside microchannels. Due to its remarkable advantages, droplet microfluidics bears significant value in an extremely wide range of area. In this review, we provide a comprehensive and in-depth insight into droplet microfluidics, covering fundamental research from microfluidic chip fabrication and droplet generation to the applications of droplets in bio(chemical) analysis and materials generation. The purpose of this review is to convey the fundamentals of droplet microfluidics, a critical analysis on its current status and challenges, and opinions on its future development. We believe this review will promote communications among biology, chemistry, physics, and materials science.

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Heat-shock transformation of Escherichia coli in nanolitre droplets formed in a capillary-composited microfluidic device

Cited by 10 publications

References 35 publications

Prospective identification of parasitic sequences in phage display screens

Prospective identification of parasitic sequences in phage display screens

Droplet microfluidics for synthetic biology

Emerging Droplet Microfluidics

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