Adhesive Tape Microfluidics with an Autofocusing Module That Incorporates CRISPR Interference: Applications to Long-Term Bacterial Antibiotic Studies
Adhesive Tape Microfluidics with an Autofocusing Module That Incorporates CRISPR Interference: Applications to Long-Term Bacterial Antibiotic Studies
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DOI：
10.1021/acssensors.9b01031
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发表时间：
2019-10-01
期刊：
影响因子：
8.9
通讯作者：
Pandey, Santosh
中科院分区：
文献类型：
--
作者：
Kong, Taejoon;Backes, Nicholas;Pandey, Santosh
在单细胞水平上研究细菌的能力提高了我们对微生物生理学和遗传学的认识，而这是使用更传统的培养方法研究大群体所无法实现的。为了改进在细胞水平上表征细菌的方法，我们开发了一种新的微流体平台，使细胞能够以浓度梯度暴露于代谢物。通过用胶带设计低成本的三维设备，并为细菌成像定制它们，我们避免了硅和聚合物微加工的复杂性。结合琼脂糖膜作为静止底物，沿着温度控制的环境室，允许在稳定生长或抑制条件下培养细菌细胞超过10小时。结合自动聚焦模块有助于在单细胞和低密度种群水平上对细菌进行不间断的高分辨率观察。我们使用微流控平台记录氨苄青霉素暴露期间大肠杆菌的形态变化，并量化抗生素的最小抑菌浓度。我们进一步证明了利用CRISPR干扰（CRISPRi）在浓度梯度中进行微调的增量基因调控的潜力。这些低成本的工程工具，当与CRISPRi等遗传方法结合使用时，应该有助于发现抗生素敏感性的新遗传决定因素，并评估抗生素在细菌培养物中的长期有效性。
The ability to study bacteria at the single cell level has advanced our insights into microbial physiology and genetics in ways not attainable by studying large populations using more traditional culturing methods. To improve methods to characterize bacteria at the cellular level, we developed a new microfluidic platform that enables cells to be exposed to metabolites in a gradient of concentrations. By designing low-cost, three-dimensional devices with adhesive tapes and tailoring them for bacterial imaging, we avoided the complexities of silicon and polymeric microfabrication. The incorporation of an agarose membrane as the resting substrate, along with a temperature-controlled environmental chamber, allows the culturing of bacterial cells for over 10 h under stable growth or inhibition conditions. Incorporation of an autofocusing module helped the uninterrupted, high-resolution observation of bacteria at the single-cell and at low density population levels. We used the microfluidic platform to record morphological changes in Escherichia coli during ampicillin exposure and to quantify the minimum inhibitory concentration of the antibiotic. We further demonstrated the potential of finely-tuned, incremental gene regulation in a concentration gradient utilizing CRISPR interference (CRISPRi). These low-cost engineering tools, when implemented in combination with genetic approaches such as CRISPRi, should prove useful to uncover new genetic determinants of antibiotic susceptibility and evaluate the long-term effectiveness of antibiotics in bacterial cultures.