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Photodegradable Hydrogels for Rapid Screening, Isolation, and Genetic Characterization of Bacteria with Rare Phenotypes.
Biomacromolecules ( IF 5.5 ) Pub Date : 2020-06-19 , DOI: 10.1021/acs.biomac.0c00543 Niloufar Fattahi 1 , Priscila A Nieves-Otero 2 , Mohammadali Masigol 1 , André J van der Vlies 1 , Reilly S Jensen 2 , Ryan R Hansen 1 , Thomas G Platt 2
Biomacromolecules ( IF 5.5 ) Pub Date : 2020-06-19 , DOI: 10.1021/acs.biomac.0c00543 Niloufar Fattahi 1 , Priscila A Nieves-Otero 2 , Mohammadali Masigol 1 , André J van der Vlies 1 , Reilly S Jensen 2 , Ryan R Hansen 1 , Thomas G Platt 2
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Screening mutant libraries (MLs) of bacteria for strains with specific phenotypes is often a slow and laborious process that requires assessment of tens of thousands of individual cell colonies after plating and culturing on solid media. In this report, we develop a three-dimensional, photodegradable hydrogel interface designed to dramatically improve the throughput of ML screening by combining high-density cell culture with precision extraction and the recovery of individual, microscale colonies for follow-up genetic and phenotypic characterization. ML populations are first added to a hydrogel precursor solution consisting of polyethylene glycol (PEG) o-nitrobenzyl diacrylate and PEG-tetrathiol macromers, where they become encapsulated into 13 μm thick hydrogel layers at a density of 90 cells/mm2, enabling parallel monitoring of 2.8 × 104 mutants per hydrogel. Encapsulated cells remain confined within the elastic matrix during culture, allowing one to track individual cells that grow into small, stable microcolonies (45 ± 4 μm in diameter) over the course of 72 h. Colonies with rare growth profiles can then be identified, extracted, and recovered from the hydrogel in a sequential manner and with minimal damage using a high-resolution, 365 nm patterned light source. The light pattern can be varied to release motile cells, cellular aggregates, or microcolonies encapsulated in protective PEG coatings. To access the benefits of this approach for ML screening, an Agrobacterium tumefaciens C58 transposon ML was screened for rare, resistant mutants able to grow in the presence of cell free culture media from Rhizobium rhizogenes K84, a well-known inhibitor of C58 cell growth. Subsequent genomic analysis of rare cells (9/28,000) that developed into microcolonies identified that seven of the resistant strains had mutations in the acc locus of the Ti plasmid. These observations are consistent with past research demonstrating that the disruption of this locus confers resistance to agrocin 84, an inhibitory molecule produced by K84. The high-throughput nature of the screen allows the A. tumefaciens genome (approximately 5.6 Mbps) to be screened to saturation in a single experimental trial, compared to hundreds of platings required by conventional plating approaches. As a miniaturized version of the gold-standard plating assay, this materials-based approach offers a simple, inexpensive, and highly translational screening technique that does not require microfluidic devices or complex liquid handling steps. The approach is readily adaptable to other applications that require isolation and study of rare or phenotypically pure cell populations.
中文翻译：
用于快速筛选、分离和遗传表征具有罕见表型的细菌的可光降解水凝胶。
筛选具有特定表型的菌株的细菌突变文库 (ML) 通常是一个缓慢而费力的过程，需要在固体培养基上铺板和培养后评估数以万计的单个细胞集落。在本报告中，我们开发了一种三维、可光降解的水凝胶界面，旨在通过将高密度细胞培养与精确提取相结合，以及为后续遗传和表型表征而回收单个微型菌落，从而显着提高 ML 筛选的通量。首先将 ML 群体添加到由聚乙二醇 (PEG)邻硝基苄基二丙烯酸酯和 PEG-四硫醇大分子单体组成的水凝胶前体溶液中，在那里它们以 90 个细胞/mm 2的密度封装在 13 μm 厚的水凝胶层中，使每个水凝胶能够并行监测 2.8 × 10 4 个突变体。封装的细胞在培养过程中仍被限制在弹性基质内，从而可以跟踪单个细胞，这些细胞在 72 小时内生长成小的、稳定的微集落（直径 45 ± 4 μm）。然后可以使用高分辨率 365 nm 图案光源以顺序方式从水凝胶中识别、提取和恢复具有稀有生长曲线的菌落，并且损伤最小。可以改变光模式以释放包裹在保护性 PEG 涂层中的运动细胞、细胞聚集体或微集落。为了获得这种方法对 ML 筛选的好处，根癌农杆菌筛选 C58 转座子 ML 以寻找能够在来自根瘤菌K84（一种众所周知的 C58 细胞生长抑制剂）的无细胞培养基存在下生长的罕见抗性突变体。随后对发育成微菌落的稀有细胞 (9/28,000) 进行基因组分析，发现其中 7 个抗性菌株在Ti 质粒的acc基因座中发生突变。这些观察结果与过去的研究一致，表明该基因座的破坏赋予了对农杆菌素 84 的抗性，农杆菌素 84 是一种由 K84 产生的抑制分子。屏幕的高通量特性允许A. tumefaciens与传统电镀方法所需的数百次电镀相比，基因组（约 5.6 Mbps）将在单个实验试验中筛选至饱和。作为金标准电镀测定的小型化版本，这种基于材料的方法提供了一种简单、廉价且高度平移的筛选技术，不需要微流体装置或复杂的液体处理步骤。该方法很容易适用于需要分离和研究稀有或表型纯细胞群的其他应用。
更新日期：2020-08-10
中文翻译：
用于快速筛选、分离和遗传表征具有罕见表型的细菌的可光降解水凝胶。
筛选具有特定表型的菌株的细菌突变文库 (ML) 通常是一个缓慢而费力的过程，需要在固体培养基上铺板和培养后评估数以万计的单个细胞集落。在本报告中，我们开发了一种三维、可光降解的水凝胶界面，旨在通过将高密度细胞培养与精确提取相结合，以及为后续遗传和表型表征而回收单个微型菌落，从而显着提高 ML 筛选的通量。首先将 ML 群体添加到由聚乙二醇 (PEG)邻硝基苄基二丙烯酸酯和 PEG-四硫醇大分子单体组成的水凝胶前体溶液中，在那里它们以 90 个细胞/mm 2的密度封装在 13 μm 厚的水凝胶层中，使每个水凝胶能够并行监测 2.8 × 10 4 个突变体。封装的细胞在培养过程中仍被限制在弹性基质内，从而可以跟踪单个细胞，这些细胞在 72 小时内生长成小的、稳定的微集落（直径 45 ± 4 μm）。然后可以使用高分辨率 365 nm 图案光源以顺序方式从水凝胶中识别、提取和恢复具有稀有生长曲线的菌落，并且损伤最小。可以改变光模式以释放包裹在保护性 PEG 涂层中的运动细胞、细胞聚集体或微集落。为了获得这种方法对 ML 筛选的好处，根癌农杆菌筛选 C58 转座子 ML 以寻找能够在来自根瘤菌K84（一种众所周知的 C58 细胞生长抑制剂）的无细胞培养基存在下生长的罕见抗性突变体。随后对发育成微菌落的稀有细胞 (9/28,000) 进行基因组分析，发现其中 7 个抗性菌株在Ti 质粒的acc基因座中发生突变。这些观察结果与过去的研究一致，表明该基因座的破坏赋予了对农杆菌素 84 的抗性，农杆菌素 84 是一种由 K84 产生的抑制分子。屏幕的高通量特性允许A. tumefaciens与传统电镀方法所需的数百次电镀相比，基因组（约 5.6 Mbps）将在单个实验试验中筛选至饱和。作为金标准电镀测定的小型化版本，这种基于材料的方法提供了一种简单、廉价且高度平移的筛选技术，不需要微流体装置或复杂的液体处理步骤。该方法很容易适用于需要分离和研究稀有或表型纯细胞群的其他应用。
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