PR-PR: Cross-Platform Laboratory Automation System

Linshiz, Gregory; Stawski, Nina; Goyal, Garima; Bi, Chen; Poust, Sean; Sharma, Monica; Mutalik, Vivek K.; Keasling, Jay D.; Hillson, Nathan J.

doi:10.1021/sb4001728

Cited by 41 publications

(52 citation statements)

References 15 publications

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“…To demonstrate the compatibility of the microfluidic heat-shock device with DNA assembly reactions, we examined the on-chip transformation of a Golden Gate assembly reaction for pProm1_BCD1-GFP expressing an ampicillin resistance gene into E. coli. DNA assembly was initially performed off-chip using the Golden Gate assembly method 7 and then 0.1 ng/μL pProm1_BCD1-GFP (1) long heat-shock at 42°C, (2) long heat-shock at 37°C, (3) quick heat-shock, (4) quick heat-shock and outgrowth, (5) quick mixing, heat-shock and outgrowth, (6) quick mixing, heat-shock, recovery and outgrowth, and (7) kanamycin resistance.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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A Droplet Microfluidic Platform for Automating Genetic Engineering

et al. 2016

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We present a water-in-oil droplet microfluidic platform for transformation, culture and expression of recombinant proteins in multiple host organisms including bacteria, yeast and fungi. The platform consists of a hybrid digital microfluidic/ channel-based droplet chip with integrated temperature control to allow complete automation and integration of plasmid addition, heat-shock transformation, addition of selection medium, culture, and protein expression. The microfluidic format permitted significant reduction in consumption (100-fold) of expensive reagents such as DNA and enzymes compared to the benchtop method. The chip contains a channel to continuously replenish oil to the culture chamber to provide a fresh supply of oxygen to the cells for long-term (∼5 days) cell culture. The flow channel also replenished oil lost to evaporation and increased the number of droplets that could be processed and cultured. The platform was validated by transforming several plasmids into Escherichia coli including plasmids containing genes for fluorescent proteins GFP, BFP and RFP; plasmids with selectable markers for ampicillin or kanamycin resistance; and a Golden Gate DNA assembly reaction. We also demonstrate the applicability of this platform for transformation in widely used eukaryotic organisms such as Saccharomyces cerevisiae and Aspergillus niger. Duration and temperatures of the microfluidic heat-shock procedures were optimized to yield transformation efficiencies comparable to those obtained by benchtop methods with a throughput up to 6 droplets/min. The proposed platform offers potential for automation of molecular biology experiments significantly reducing cost, time and variability while improving throughput.

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Section: ■ Results and Discussionmentioning

confidence: 99%

“…5 Various molecular biology steps have been performed using microfluidic systems including synthesis of DNA, 6,7 cell sorting, 8 single cell analysis, 9 and cellular assays. 10 Microfluidic devices have also been developed to provide gene delivery to cells by chemical heat-shock and electroporation.…”

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A Droplet Microfluidic Platform for Automating Genetic Engineering

et al. 2016

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“…Golden Gate parts can be generated by PCR or by gene synthesis 27,28 , but in order to minimize errors in assembly the basic assembly parts are usually first placed in 'entry vectors' and then digested and ligated into 'destination vectors' (Figure 1). To enable using Golden Gate for hierarchical assembly, a variety of physical standards have been developed that adopt a tiered assembly approach: in the first tier genes are assembled from their elementary parts (promoters, open reading frames, terminators, etc.…”

Section: Endonuclease-mediated Assemblymentioning

confidence: 99%

“…Golden Gate cloning is based on these principles, and has gained significant popularity especially in publically-available kits where many parts and/or highly repetitive sequences need to be assembled in one go [24][25][26] . One of the main challenges in assembly with Golden Gate is defining the position of the DNA parts within the final construct, which depends on the sequences of the short overhangs generated by digestion.Golden Gate parts can be generated by PCR or by gene synthesis 27,28 , but in order to minimize errors in assembly the basic assembly parts are usually first placed in 'entry vectors' and then digested and ligated into 'destination vectors' (Figure 1). To enable using Golden Gate for hierarchical assembly, a variety of physical standards have been developed that adopt a tiered assembly approach: in the first tier genes are assembled from their elementary parts (promoters, open reading frames, terminators, etc.…”

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confidence: 99%

Bricks and blueprints: methods and standards for DNA assembly

Casini

Storch

Baldwin

et al. 2015

Nat Rev Mol Cell Biol

252

186

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PrefaceDNA assembly is a key part of constructing gene expression systems and even whole chromosomes. In the past decade a plethora of powerful new DNA assembly methods including Gibson assembly, Golden Gate and LCR have been developed. In this Innovation article we discuss these methods and standards such as MoClo, GoldenBraid, MODAL and PaperClip, which have been developed to facilitate a streamlined assembly workflow, aid material exchange, and the creation of modular, reusable DNA parts. IntroductionOur capacity to cut and paste DNA from different sources and to assemble it into gene constructs has been one of the key drivers of biological research and biotechnology over the past four decades. However, despite countless advances in molecular biology, the assembly of DNA parts into new constructs remains a craft that is both time consuming and unpredictable. The decreasing costs of gene synthesis promises to alleviate these limitations by providing custom-made double-stranded DNA fragments typically between 200 and 2000 bp in length 1 . Nonetheless, gene synthesis does not eliminate the need for DNA assembly, which remains necessary for the production of constructs beyond one kilobase in size, both in research labs and at gene synthesis companies. DNA assembly also enables carrying out projects with more complex experimental needs and is especially valuable for building diverse plasmid libraries and creating multicomponent systems, and has even been used to construct synthetic cells 2 .Addressing the limitations of DNA assembly methods has been one of the key goals of synthetic biology, a scientific discipline focused on the construction and testing of new or redesigned versions of genes, gene networks, pathways and cells 3,4 . In order to tackle projects of increasing scale and complexity, researchers have invested significant effort into developing new tools for DNA assembly and into matching them with improved, lower-cost gene synthesis (for reviewes on gene synthesis see REFS. 1,5 1,5 ), as well as a suite of important new tools for genome editing (Box 1). With these combined advances the field is now at a point where gene synthesis and DNA assembly can empower even undergraduate students to construct entire eukaryotic chromosomes 6 . This acceleration in the scale of DNA assembly enables construction projects too complex to be drawn out on the back of an envelope, which instead require an engineering approach. In the past decade important 1 assembly methods such as Gibson assembly and Golden Gate have been developed 7,8 , which define new protocols for joining together DNA parts. Alongside these methods, researchers have also developed various physical standards such as MODAL (Modular Overlap-Directed Assembly with Linkers) 9 and MoClo (Modular Cloning system) 12 that define rules for the format of DNA parts that can be used with them. These physical standards facilitate the re-use of parts between experiments, exchange of parts between research groups and importantly provide modularity in construction. ...

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“…Currently, instruments for online sampling, sample preparation, and transfer to analytical devices are being integrated into cell culture systems 243 . Several automation programming platforms have been developed that allow for cross-platform liquid handling using microfluidic devices 244,245 . Such systems can drastically decrease the time requirement and labor intensity on the sampling actions of mL-scale systems.…”

Section: Scale-down Verificationmentioning

confidence: 99%

Scale-down of CHO cell fed-batch cultures

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PR-PR: Cross-Platform Laboratory Automation System

Cited by 41 publications

References 15 publications

A Droplet Microfluidic Platform for Automating Genetic Engineering

A Droplet Microfluidic Platform for Automating Genetic Engineering

Bricks and blueprints: methods and standards for DNA assembly

Scale-down of CHO cell fed-batch cultures

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