Automated, High-Throughput Protoplast Transfection for Gene Editing and Transgene Expression Studies

Rigoulot, Stephen B.; Barco, Brenden; Zhang, Yingxiao; Zhang, Chengjin; Meier, Kerry A.; Moore, Matthew T.; Fabish, Jonathan; Whinna, Rachel; Park, Jeongmoo; Seaberry, Erin M.; Gopalan, Aditya; Dong, Shiqi; Chen, Zhongying; Que, Qiudeng

doi:10.1007/978-1-0716-3131-7_9

Cited by 3 publications

(3 citation statements)

References 32 publications

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“…Synthetic promoter libraries used in ML and DL architecture are not only of a large size but they need to indicate a sufficient dynamic range, gradient strength, and clear sequence profiles [46]. However, the large scale of the applied libraries requires the more broad application of high-throughput analytical tool methods, such as high-capacity protoplast transformation, combined with a fluorescence-inducing laser projector (FLIP) platform equipped with an ultra-low-noise camera; this has been used to discriminate between numerous fluorescent signatures under different stress conditions [253][254][255].…”

Section: Discussionmentioning

confidence: 99%

“…Advanced ML and DL algorithms produce a large quantity of results which should be verified and validated experimentally. To meet such analytical requirements, highthroughput, automated methods of protoplast transformation were developed [253,254]. Moreover, the phenotypic properties of transformed protoplasts, expressing fluorescent proteins (FPs) could be studied by the fluorescence-inducing laser projector (FILP)-supported by an ultra-low-noise camera to resolve the individual FP's patterns and to phenotype transgenic plants [255].…”

Section: Machine Learning and Deep Learning Support To Synthetic Prom...mentioning

confidence: 99%

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Plant Synthetic Promoters

Szymczyk,

Majewska

2024

Applied Sciences

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This article examines the structure and functions of the plant synthetic promoters frequently used to precisely regulate complex regulatory routes. It details the composition of native promoters and their interacting proteins to provide a better understanding of the tasks associated with synthetic promoter development. The production of synthetic promoters is performed by relatively small libraries produced generally by basic molecular or genetic engineering methods such as cis-element shuffling or domain swapping. The article also describes the preparation of large-scale libraries supported by synthetic DNA fragments, directed evolution, and machine or deep-learning methodologies. The broader application of novel, synthetic promoters reduces the prevalence of homology-based gene silencing or improves the stability of transgenes. A particularly interesting group of synthetic promoters are bidirectional forms, which can enable the expression of up to eight genes by one regulatory element. The introduction and controlled expression of several genes after one transgenic event strongly decreases the frequency of such problems as complex segregation patterns and the random integration of multiple transgenes. These complications are commonly observed during the transgenic crop development enabled by traditional, multistep transformation using genetic constructs containing a single gene. As previously tested DNA promoter fragments demonstrate low complexity and homology, their abundance can be increased by using orthogonal expression systems composed of synthetic promoters and trans-factors that do not occur in nature or arise from different species. Their structure, functions, and applications are rendered in the article. Among them are presented orthogonal systems based on transcription activator-like effectors (dTALEs), synthetic dTALE activated promoters (STAPs) and dCas9-dependent artificial trans-factors (ATFs). Synthetic plant promoters are valuable tools for providing precise spatiotemporal regulation and introducing logic gates into the complex genetic traits that are important for basic research studies and their application in crop plant development. Precisely regulated metabolic routes are less prone to undesirable feedback regulation and energy waste, thus improving the efficiency of transgenic crops.

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Section: Discussionmentioning

confidence: 99%

Section: Machine Learning and Deep Learning Support To Synthetic Prom...mentioning

confidence: 99%

Plant Synthetic Promoters

Szymczyk,

Majewska

2024

Applied Sciences

View full text Add to dashboard Cite

show abstract

“…However, the scale of the applied libraries results in problems with prompt and accurate analysis of the obtained variant properties. A promising solution could be the more broad application of a fluorescence-inducing laser projector (FLIP) platform equipped with an ultra-low-noise camera, which enabled researchers to discriminate between numerous fluorescent signatures under different stress conditions [261][262][263].…”

Section: Discussionmentioning

confidence: 99%

Plant Synthetic Promoters

Szymczyk,

Majewska

2024

Preprint

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The article features the structure and functions of plant synthetic promoters frequently exercised to precisely regulate complex regulatory routes. The composition of plant native promoters together with interacting proteins is presented to provide a better understanding of tasks associated with synthetic promoter development. The production of synthetic promoters is conferred on relatively small libraries produced generally by basic molecular or genetic engineering methods such as cis-element shuffling or domain swapping. Moreover, the preparation of large-scale libraries supported by synthetic DNA fragments, directed evolution, and machine or deep learning methodologies is presented. A particularly interesting group of synthetic promoters are bidirectional forms that enable the putative expression of up to 6–8 genes by one regulatory element. The introduction and controlled expression of several genes after one transgenic event strongly decreases the frequency of such problems as complex segregation patterns and random integration of multiple transgenes. These complications are commonly observed during transgenic crop development through traditional, multistep transformation by genetic constructs containing a single gene. Another path to solving problems associated with the low complexity and homology of already tested DNA fragments is through orthogonal expression systems composed of synthetic promoters and trans-factors that do not occur in nature or arise from different species. Their structure, functions, and applications are rendered in the article.

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Enhancing lipid production in plant cells through high-throughput genome editing and phenotyping via a scalable automated pipeline

Dong,

Croslow,

Lane

et al. 2024

Preprint

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Plant bioengineering is a time-consuming and labor-intensive process, with no guarantee of achieving the desired trait. Here we report a fast, automated, scalable, high-throughput pipeline for plant bioengineering (FAST-PB). FAST-PB achieves gene cloning, genome editing, and product characterization by integrating automated biofoundry engineering of callus and protoplast cells with single cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We first demonstrate that FAST-PB can streamline the Golden Gate cloning process, with the capacity to construct 96 vectors in parallel. To prove the concept, using FAST-PB, we first found that PEG2050 significantly increases transfection efficiency by over 45%. To validate the pipeline, we established a reporter-gene-free method for CRISPR editing via mutation ofHCF136, affecting cellular chlorophyll fluorescence. Next, we applied this pipeline for lipid production and found that diverse lipids were significantly enhanced up to sixfold through introducing multi-gene cassettes via CRISPR activation, and regenerated plant using this platform. Lastly, we harnessed FAST-PB to achieve high-throughput single-cell lipid profiling through the integration of MALDI-MS with the biofoundry, and differentiated engineered and unengineered cells using the single-cell lipidomics. These innovations massively increase the throughput of synthetic biology, genome editing, and metabolic engineering, and change what is possibly using single-cell metabolomics in plants.

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Automated, High-Throughput Protoplast Transfection for Gene Editing and Transgene Expression Studies

Cited by 3 publications

References 32 publications

Plant Synthetic Promoters

Plant Synthetic Promoters

Plant Synthetic Promoters

Enhancing lipid production in plant cells through high-throughput genome editing and phenotyping via a scalable automated pipeline

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