Logic Circuits Based on 2A Peptide Sequences in the Yeast Saccharomyces cerevisiae,ACS Synthetic Biology

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Logic Circuits Based on 2A Peptide Sequences in the Yeast Saccharomyces cerevisiae
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2022-12-22 , DOI: 10.1021/acssynbio.2c00506
Xuekun Wang ₁ , Ximing Tian ₁ , Mario Andrea Marchisio ₁

Affiliation

Gene digital circuits are the subject of many studies in Synthetic Biology due to their various applications from pollutant detection to medical diagnostics and biocomputing. Complex logic functions are calculated via small genetic components that mimic Boolean gates, i.e., they implement basic logic operations. Gates interact by exchanging proteins or noncoding RNAs. To carry out logic operations in the yeast Saccharomyces cerevisiae, we chose three bacterial repressors commonly used for proofs of concept in Synthetic Biology, namely, TetR, LexA, and LacI. We coexpressed them via synthetic polycistronic cassettes based on 2A peptide sequences. Our initial results highlighted the successful application of four 2A peptides─from Equine rhinitis B virus-1 (ERBV-1 2A), Operophtera brumata cypovirus 18 (OpbuCPV18 2A), Ljungan virus (LV2A), and Thosea asigna virus (T2A)─to the construction of single and two-input Boolean gates. In order to improve protein coexpression, we modified the original 2A peptides with the addition of the glycine-serine-glycine (GSG) prefix or by using two different 2As sequences in tandem. Remarkably, we finally realized a well-working tri-cistronic vector that carried LexA-HBD(hER), TetR, and LacI separated, in the order, by GSG-T2A and ERBV-1 2A. This plasmid led to the implementation of three-input circuits containing AND and OR gates. Taken together, polycistronic constructs simplify the cloning and coexpression of multiple proteins with a dramatic reduction in the complexity of gene digital circuits.

中文翻译：

基于酿酒酵母中 2A 肽序列的逻辑电路

由于从污染物检测到医学诊断和生物计算的各种应用，基因数字电路是合成生物学中许多研究的主题。复杂的逻辑函数是通过模拟布尔门的小型遗传组件计算的，即它们实现基本的逻辑运算。盖茨通过交换蛋白质或非编码 RNA 相互作用。为了在酿酒酵母中进行逻辑运算，我们选择了合成生物学中常用于概念验证的三种细菌阻遏物，即 TetR、LexA 和 LacI。我们通过基于 2A 肽序列的合成多顺反子盒共表达它们。我们的初步结果突出了四种 2A 肽的成功应用─来自马鼻炎 B病毒 1 (ERBV-1 2A)，Operophtera brumata cypovirus 18 (OpbuCPV18 2A)、Ljungan病毒 (LV2A) 和Thesea asigna病毒 (T2A)──构建单输入和双输入布尔门。为了改善蛋白质共表达，我们通过添加甘氨酸-丝氨酸-甘氨酸 (GSG) 前缀或串联使用两个不同的 2As 序列来修饰原始 2A 肽。值得注意的是，我们最终实现了一个运行良好的三顺反子载体，该载体携带按 GSG-T2A 和 ERBV-1 2A 顺序分开的 LexA-HBD(hER)、TetR 和 LacI。该质粒导致了包含“与”门和“或”门的三输入电路的实现。总之，多顺反子结构简化了多种蛋白质的克隆和共表达，同时显着降低了基因数字电路的复杂性。

更新日期：2022-12-22

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