Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Department of Bioengineering and Department of Electrical and Computer Engineering, Northeastern University, Boston, Massachusetts, USA.
Nat Biotechnol. 2018 Apr;36(4):352-358. doi: 10.1038/nbt.4111. Epub 2018 Mar 19.
The internal environment of growing cells is variable and dynamic, making it difficult to introduce reliable parts, such as promoters, for genetic engineering. Here, we applied control-theoretic ideas to design promoters that maintained constant levels of expression at any copy number. Theory predicts that independence to copy number can be achieved by using an incoherent feedforward loop (iFFL) if the negative regulation is perfectly non-cooperative. We engineered iFFLs into Escherichia coli promoters using transcription-activator-like effectors (TALEs). These promoters had near-identical expression in different genome locations and plasmids, even when their copy number was perturbed by genomic mutations or changes in growth medium composition. We applied the stabilized promoters to show that a three-gene metabolic pathway to produce deoxychromoviridans could retain function without re-tuning when the stabilized-promoter-driven genes were moved from a plasmid into the genome.
细胞生长的内部环境是多变和动态的，这使得难以引入可靠的部分，如启动子，用于基因工程。在这里，我们应用控制理论的思想来设计启动子，使其在任何拷贝数下都能保持恒定的表达水平。理论预测，如果负反馈是完全非合作的，那么通过使用非相干前馈环（iFFL）可以实现对拷贝数的独立性。我们使用转录激活子样效应物（TALEs）将 iFFL 工程化到大肠杆菌启动子中。这些启动子在不同的基因组位置和质粒中具有几乎相同的表达，即使它们的拷贝数受到基因组突变或生长介质组成变化的干扰。我们应用稳定化启动子来表明，生产脱氧胆色原杆菌的三基因代谢途径在不重新调整的情况下保留功能，当稳定化启动子驱动的基因从质粒转移到基因组时。
