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Design and Selection of a Synthetic Feedback Loop for Optimizing Biofuel Tolerance
ACS Synthetic Biology ( IF 4.7 ) Pub Date : 2017-10-12 00:00:00 , DOI: 10.1021/acssynbio.7b00260
Yik Siu 1 , Jesse Fenno 1 , Jessica M. Lindle 1 , Mary J. Dunlop 1, 2
Affiliation  

Feedback control allows cells to dynamically sense and respond to environmental changes. However, synthetic controller designs can be challenging because of implementation issues, such as determining optimal expression levels for circuit components within a feedback loop. Here, we addressed this by coupling rational design with selection to engineer a synthetic feedback circuit to optimize tolerance of Escherichia coli to the biojet fuel pinene. E. coli can be engineered to produce pinene, but it is toxic to cells. Efflux pumps, such as the AcrAB-TolC pump, can improve tolerance, but pump expression impacts growth. To address this, we used feedback to dynamically regulate pump expression in response to stress. We developed a library with thousands of synthetic circuit variants and subjected it to three types of pinene treatment (none, constant, and varying pinene). We were able to select for strains that were biofuel tolerant without a significant growth cost in the absence of biofuel. Using next-generation sequencing, we found common characteristics in the designs and identified controllers that dramatically improved biofuel tolerance.

中文翻译:

优化生物燃料耐受性的合成反馈回路的设计与选择

反馈控制使细胞能够动态感应并响应环境变化。然而,由于实现问题,例如确定反馈回路内电路组件的最佳表达水平,合成控制器设计可能具有挑战性。在这里,我们通过将合理的设计与选择相结合来解决这一问题,从而设计出一个合成反馈电路来优化大肠杆菌对生物喷气燃料pin烯的耐受性。大肠杆菌可以被工程化生产pin烯,但是对细胞有毒。外排泵,例如AcrAB-TolC泵,可以提高耐受性,但泵的形状会影响其生长。为了解决这个问题,我们使用反馈来响应压力来动态调节泵的表达。我们开发了一个库,其中包含数千个合成电路变体,并对其进行了三种类型的pin烯处理(无,恒定和可变的pin烯)。在没有生物燃料的情况下,我们能够选择具有生物燃料耐受性的菌株,而不会产生显着的增长成本。使用下一代测序,我们在设计中发现了共同特征,并确定了可显着提高生物燃料耐受性的控制器。
更新日期:2017-10-12
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