I-Corps: Kytopen: Expanding the language of biology with pulsed electric fields

解决人类面临的许多最紧迫的挑战，包括对替代燃料的需求，提高石油采收率，甚至治疗癌症，都可能涉及到改造目前人类无法企及的众多细菌。有许多类型的细菌可以在实验室中生长，但它们对人们生活的潜在影响还没有意识到。需要新的工具来解锁细菌吗？解决人类感兴趣的许多挑战的真正潜力。创造工具来加速发现新的癌症治疗方法、替代燃料或低成本生物材料，将对人类生活产生重大影响。人口增长和寿命延长对环境的要求越来越高，推动了对替代燃料、食品和药品来源的需求。细菌作为创新的源泉一直被忽视；然而，他们已经有了内部机制来帮助以快速有效的方式完成工业过程。脉冲电场（即电穿孔）可用于将遗传物质输送到包括细菌在内的许多类型的细胞中。本提案的目标是大大扩展脉冲电场在细菌上的成功应用，并使发现基因工程和合成生物学中的新应用成为可能。合成生物学的一个主要限制是无法将遗传物质整合到许多细菌中，这是由于在保持高细胞活力的同时渗透细胞包膜的挑战。该团队的目标是将开发基因操纵细菌的工具所需的时间从几个月甚至几年缩短到几天。这个I-Corps团队开发了一种概念验证型微流体装置，可以在单个实验中表征特定实验条件下细菌电穿孔的临界电场。该团队正在设计一种技术，可以在一天内测试数千种独特的条件，跨越整个参数空间。这将使用一个微流控平台，或多个并行平台，在不同的实验条件下测试小份额的细胞。因此，所提出的微流体系统能够在单个实验中量化关键的电穿孔参数，否则需要数月的实验。提出的创新将带来两个重大进步：1)快速确定高产量电穿孔条件的能力；2)利用迄今为止难以处理或难以基因操纵的10,000多种细菌菌株的能力。

The potential to solve many of mankind's most pressing challenges including the needs for alternative fuels, enhancing oil recovery, and even treating cancer could involve engineering the numerous bacteria currently beyond one's reach. There are many types of bacteria that can be grown in the lab but their potential impact on people's lives has not been realized. New tools are needed to unlock bacteria?s true potential to solve many challenges of interest to mankind. Creating tools to accelerate discovery of new cancer treatments, alternative fuels, or low cost biomaterials will significantly impact human life. Population growth and increasing lifespans are putting ever increasing demands on the environment, driving need for alternative sources of fuels, food, and medicines. Bacteria have been a bit neglected as sources for innovation; however, they already have the internal machinery to help do industrial processes in a fast and efficient manner.Pulsed electric fields (i.e. electroporation) can be used to deliver genetic material into many types of cells including bacteria. The goal of this proposal is to substantially expand the successful application of pulsed electric fields to bacteria and enable discovery of new applications in genetic engineering and synthetic biology. A major limitation of synthetic biology is the inability to incorporate genetic material into many bacteria due to the challenge of permeating the cell envelope while maintaining high cell viability. This team's goal is to reduce the time required to develop tools to genetically manipulate bacteria from months or even years to days. This I-Corps team has developed a proof-of-concept microfluidic device to enable the characterization of the critical electric field for bacterial electroporation under specific experimental conditions in a single experiment. The team is devising a technique to test several thousand unique conditions within a single day, spanning the entire parametric space. This will be done using a microfluidic platform, or several in parallel, to test small aliquots of cells at varying experimental conditions. Therefore, the proposed microfluidic system enables quantification of the critical electroporation parameters in a single experiment, which would otherwise require months of experimentation. The proposed innovation will allow two significant advancements: 1) the ability to quickly determine high yield electroporation conditions and 2) the ability to utilize many of the more than 10,000 bacterial strains that have been heretofore intractable or difficult to genetically manipulate.