Genetic circuit performance under conditions relevant for industrial bioreactors.
Genetic circuit performance under conditions relevant for industrial bioreactors.
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DOI：
10.1021/sb3000832
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发表时间：
2012-11-16
影响因子：
4.7
通讯作者：
Voigt, Christopher A.
中科院分区：
文献类型：
--
作者：
Moser, Felix;Broers, Nicolette J.;Hartmans, Sybe;Tamsir, Alvin;Kerkman, Richard;Roubos, Johannes A.;Bovenberg, Roel;Voigt, Christopher A.
关键词：
合成遗传程序有望在工业过程中实现新的应用。对于这样的应用，组成程序的基因电路将需要在变化和复杂的环境中保持保真度。在这项工作中，我们报告了在工业相关条件下大肠杆菌中两种合成电路的性能，包括培养基，菌株和生长速率的选择。我们测试和比较两个转录电路：与门和或非门。在大肠coliDH10B中，与门在基本培养基中失活，通过补充培养基并将与门转移到工业菌株E. coliDS68637，其中在基本培养基中观察到正常功能。相比之下，NOR门对培养基组成是稳健的，并且在两种菌株中功能相似。在早期放大的三个阶段评估与门：100 ml摇瓶实验，1 ml MTP微反应器和10 L生物反应器。组成型产生GFP报告基因的参考质粒用于比较不同条件下的电路性能。与门的功能在每个刻度上都是定量不同的。在从指数进料速率转变为恒定进料速率后，发酵后期的产量恶化，这导致资源快速消耗和生长速率的变化。此外，与门的一个输出状态在生物反应器中失败，有效地使其仅响应于单个输入。最后，携带AND门的细胞显示出相当少的生物质积累。总的来说，这些结果突出了挑战，并提出了改进的策略，用于开发和表征在发酵过程中可靠发挥作用的遗传电路。
Synthetic genetic programs promise to enable novel applications in industrial processes. For such applications, the genetic circuits that compose programs will require fidelity in varying and complex environments. In this work, we report the performance of two synthetic circuits in Escherichia coli under industrially relevant conditions, including the selection of media, strain, and growth rate. We test and compare two transcriptional circuits: an AND and a NOR gate. In E. coli DH10B, the AND gate is inactive in minimal media; activity can be rescued by supplementing the media and transferring the gate into the industrial strain E. coli DS68637 where normal function is observed in minimal media. In contrast, the NOR gate is robust to media composition and functions similarly in both strains. The AND gate is evaluated at three stages of early scale-up: 100 ml shake-flask experiments, a 1 ml MTP microreactor, and a 10 L bioreactor. A reference plasmid that constitutively produces a GFP reporter is used to make comparisons of circuit performance across conditions. The AND gate function is quantitatively different at each scale. The output deteriorates late in fermentation after the shift from exponential to constant feed rates, which induces rapid resource depletion and changes in growth rate. In addition, one of the output states of the AND gate failed in the bioreactor, effectively making it only responsive to a single input. Finally, cells carrying the AND gate show considerably less accumulation of biomass. Overall, these results highlight challenges and suggest modified strategies for developing and characterizing genetic circuits that function reliably during fermentation.
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影响因子：
3
作者：
Baker, David;Group, B. I. O. F. A. B.;Weiss, Ron
通讯作者：
Weiss, Ron
影响因子：
4.1
作者：
Enfors, SO;Jahic, M;Manelius, Å
通讯作者：
Manelius, Å
影响因子：
46.9
作者：
Canton, Barry;Labno, Anna;Endy, Drew
通讯作者：
Endy, Drew
DOI：
10.1073/pnas.120163297
发表时间：
2000-06-06
影响因子：
11.1
作者：
Datsenko, KA;Wanner, BL
通讯作者：
Wanner, BL
影响因子：
2.7
作者：
Han, L;Enfors, SO;Häggström, L
通讯作者：
Häggström, L