Acoustofluidic medium exchange for preparation of electrocompetent bacteria using channel wall trapping.
Acoustofluidic medium exchange for preparation of electrocompetent bacteria using channel wall trapping.
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利用声流媒体交换技术制备电感受态细菌。
DOI：
10.1039/d1lc00406a
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发表时间：
2021-11-09
期刊：
影响因子：
6.1
通讯作者：
Dual J
中科院分区：
文献类型：
--
作者：
Gerlt MS;Ruppen P;Leuthner M;Panke S;Dual J
将工艺步骤全面集成到合成生物学工作流程的小型化版本中，仍然是生物系统设计自动化的关键任务。然而，这些工艺步骤中的每一个都对环境条件有特定的要求，特别是对周围流体的组成，这使得集成变得繁琐。例如，转化，即通过将外源遗传物质(如DNA)输送到细胞质中对细菌进行重新编程，是分子工程和现代生物技术中的一个关键过程。转化通常是通过电穿孔进行的，即在低电导率环境中使用电击在膜上制造孔。然而，用于电穿孔的细胞准备可能会很麻烦，因为它需要将生长介质(高电导率)更换为低电导率介质，通常通过多个时间密集型离心步骤来执行。为了简化和小型化这一步骤，我们开发了一种声流控设备，能够非侵入性地捕获大肠杆菌用于随后的介质交换，由于有害的声流效应，这在声流控设备中是具有挑战性的。通过改进的蚀刻工艺，我们能够在两个微流控通道之间制造出一层薄壁，当激励时，它可以产生补充声辐射力的流动场，因此可以用于捕获细菌。我们的新设计以10μL−1的流速强劲地捕获大肠杆菌，洗涤捕获的细胞后细胞回收率为47±3%。为了验证介质交换装置的性能，我们在标准转化程序中测试了回收细胞的电活性，发现每μg质粒DNA的转化效率为8×105CFU。我们的设备是一种基于离心法的小批量替代方法，并为大量微生物和分子工程方案的微型化打开了大门。将工艺步骤全面集成到合成生物学工作流程的小型化版本中，仍然是生物系统设计自动化的关键任务。我们提出了一种声流控芯片，能够自动进行细菌的介质交换。
Comprehensive integration of process steps into a miniaturised version of synthetic biology workflows remains a crucial task in automating the design of biosystems. However, each of these process steps has specific demands with respect to the environmental conditions, including in particular the composition of the surrounding fluid, which makes integration cumbersome. As a case in point, transformation, i.e. reprogramming of bacteria by delivering exogenous genetic material (such as DNA) into the cytoplasm, is a key process in molecular engineering and modern biotechnology in general. Transformation is often performed by electroporation, i.e. creating pores in the membrane using electric shocks in a low conductivity environment. However, cell preparation for electroporation can be cumbersome as it requires the exchange of growth medium (high-conductivity) for low-conductivity medium, typically performed via multiple time-intensive centrifugation steps. To simplify and miniaturise this step, we developed an acoustofluidic device capable of trapping the bacterium Escherichia coli non-invasively for subsequent exchange of medium, which is challenging in acoustofluidic devices due to detrimental acoustic streaming effects. With an improved etching process, we were able to produce a thin wall between two microfluidic channels, which, upon excitation, can generate streaming fields that complement the acoustic radiation force and therefore can be utilised for trapping of bacteria. Our novel design robustly traps Escherichia coli at a flow rate of 10 μL min−1 and has a cell recovery performance of 47 ± 3% after washing the trapped cells. To verify that the performance of the medium exchange device is sufficient, we tested the electrocompetence of the recovered cells in a standard transformation procedure and found a transformation efficiency of 8 × 105 CFU per μg of plasmid DNA. Our device is a low-volume alternative to centrifugation-based methods and opens the door for miniaturisation of a plethora of microbiological and molecular engineering protocols. Comprehensive integration of process steps into a miniaturised version of synthetic biology workflows remains a crucial task in automating the design of biosystems. We present an acoustofluidic chip, capable of automated medium exchange of bacteria.
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影响因子：
3.4
作者：
Gerlt MS;Läubli NF;Manser M;Nelson BJ;Dual J
通讯作者：
Dual J
影响因子：
4.3
作者：
Gautam GP;Gurung R;Fencl FA;Piyasena ME
通讯作者：
Piyasena ME
影响因子：
2.4
作者：
Glynne-Jones, P.;Boltryk, R. J.;Baclet, P.
通讯作者：
Baclet, P.
影响因子：
3.2
作者：
Cetin, Barbaros;Ozer, Mehmet Bulent;Buyukkocak, Suleyman
通讯作者：
Buyukkocak, Suleyman
影响因子：
4.2
作者：
Fong, Erika J.;Johnston, Amanda C.;Shusteff, Maxim
通讯作者：
Shusteff, Maxim