Dataset Information

Multi-dimensional studies of synthetic genetic promoters enabled by microfluidic impact printing.

ABSTRACT: Natural genetic promoters are regulated by multiple cis and trans regulatory factors. For quantitative studies of these promoters, the concentration of only a single factor is typically varied to obtain the dose response or transfer function of the promoters with respect to the factor. Such design of experiments has limited our ability to understand quantitative, combinatorial interactions between multiple regulatory factors at promoters. This limitation is primarily due to the intractable number of experimental combinations that arise from multifactorial design of experiments. To overcome this major limitation, we integrate impact printing and cell-free systems to enable multi-dimensional studies of genetic promoters. We first present a gradient printing system which comprises parallel piezoelectric cantilever beams as a scalable actuator array to generate droplets with tunable volumes in the range of 100 pL-10 nL, which facilitates highly accurate direct dilutions in the range of 1-10?000-fold in a 1 ?L drop. Next, we apply this technology to study interactions between three regulatory factors at a synthetic genetic promoter. Finally, a mathematical model of gene regulatory modules is established using the multi-parametric and multi-dimensional data. Our work creates a new frontier in the use of cell-free systems and droplet printing for multi-dimensional studies of synthetic genetic constructs.

SUBMITTER: Fan J

PROVIDER: S-EPMC5572659 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Multi-dimensional studies of synthetic genetic promoters enabled by microfluidic impact printing.

Fan Jinzhen J Villarreal Fernando F Weyers Brent B Ding Yunfeng Y Tseng Kuo Hao KH Li Jiannan J Li Baoqing B Tan Cheemeng C Pan Tingrui T

Lab on a chip 20170601 13

Natural genetic promoters are regulated by multiple cis and trans regulatory factors. For quantitative studies of these promoters, the concentration of only a single factor is typically varied to obtain the dose response or transfer function of the promoters with respect to the factor. Such design of experiments has limited our ability to understand quantitative, combinatorial interactions between multiple regulatory factors at promoters. This limitation is primarily due to the intractable numbe ...[more]

PMID: 28613297

Similar Datasets

Project description:Electro-transfection is an essential workhorse tool for regulating cellular responses and engineering cellular materials in tissue engineering. However, most of the existing approaches are only focused on cell suspensions in vitro, which fails to mimic an in vivo tissue microenvironment regarding the 3D electric field distribution and mass transport in a biological matrix. However, building a 3D electro-transfection system that is compatible with 3D cell culture for mimicking the in vivo tissue microenvironment is challenging, due to the substantial difficulties in control of the 3D electric field distribution as well as the cellular growth. To address such challenges, we introduce a novel 3D micro-assembly strategy assisted by 3D printing, which enables the molding of 3D microstructures as LEGO® parts from 3D-printed molds. The molded PDMS LEGO® bricks are then assembled into a 3D-cell culture chamber interconnected with vertical and horizontal perfusion microchannels as a 3D channel network. Such a 3D perfusion microchannel network is unattainable by direct 3D printing or other microfabrication approaches, which can facilitate the highly-efficient exchange of nutrition and waste for 3D cell growth. Four flat electrodes are mounted into the 3D culture chamber via a 3D-printed holder and controlled by a programmable power sequencer for multi-directional electric frequency scanning (3D ?-electro-transfection). This multi-directional scanning not only can create transient pores all over the cell membrane, but also can generate local oscillation for enhancing mass transport and improving cell transfection efficiency. As a proof-of-concept, we electro-delivered the pAcGFP1-C1 vector to 3D cultured HeLa cells within peptide hydrogel scaffolding. The expressed GFP level from transfected HeLa cells reflects the transfection efficiency. We found two key parameters including electric field strength and plasmid concentration playing more important roles than the pulse duration and duty cycles. The results showed an effective transfection efficiency of ?15% with ?85% cell viability, which is 3-fold higher compared to that of the conventional benchtop 3D cell electro-transfection. This 3D ?-electrotransfection system was further used for genetically editing 3D-cultured Hek-293 cells via direct delivery of CRISPR/Cas9 plasmid which showed successful transfection with GFP expressed in the cytoplasm as the reporter. The 3D-printing enabled micro-assembly allows facile creation of a novel 3D culture system for electro-transfection, which can be employed for versatile gene delivery and cellular engineering, as well as building in vivo like tissue models for fundamentally studying cellular regulation mechanisms at the molecular level.

Dataset Information

Multi-dimensional studies of synthetic genetic promoters enabled by microfluidic impact printing.

Publications

Multi-dimensional studies of synthetic genetic promoters enabled by microfluidic impact printing.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets