Ratiometric Gas Reporting: A Nondisruptive Approach To Monitor Gene Expression in Soils
Ratiometric Gas Reporting: A Nondisruptive Approach To Monitor Gene Expression in Soils
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DOI：
10.1021/acssynbio.7b00405
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发表时间：
2018-03-01
影响因子：
4.7
通讯作者：
Silberg, Jonathan J.
中科院分区：
文献类型：
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作者：
Cheng, Hsiao-Ying;Masiello, Caroline A.;Silberg, Jonathan J.
荧光蛋白是普遍存在的工具，用于监测自然和合成遗传电路的动态功能。然而，这些视觉报告只能在透明的环境中使用，这一限制使得在许多基质（如土壤和沉积物）中进行基因表达的非破坏性测量变得复杂。我们描述了一种新的比率气体报告方法，用于在难以成像的环境矩阵中无损监测基因表达。该方法通过乙烯形成酶连续合成C2H4以提供活细胞数的信息，通过将甲基卤化物转移酶基因置于条件启动子的控制下有条件地合成CH3Br。我们表明，比率气体报告可以创建大肠杆菌生物传感器，报告用于革兰氏阴性菌群体感应的酰基高丝氨酸内酯（AHL）自诱导剂。使用这些生物传感器，我们发现农业土壤将长链AHL的生物可利用浓度降低了100倍。我们还证明了这些生物传感器可以在土壤中对豆科根瘤菌合成并被苏云金芽孢杆菌降解的ahl进行无损监测。最后，我们证明了这种新的报告方法可以用于希瓦氏菌，一种生活在沉积物中的细菌。
Fluorescent proteins are ubiquitous tools that are used to monitor the dynamic functions of natural and synthetic genetic circuits. However, these visual reporters can only be used in transparent settings, a limitation that complicates nondisruptive measurements of gene expression within many matrices, such as soils and sediments. We describe a new ratiometric gas reporting method for nondisruptively monitoring gene expression within hard-to-image environmental matrices. With this approach, C2H4 is continuously synthesized by ethylene forming enzyme to provide information on viable cell number, and CH3Br is conditionally synthesized by placing a methyl halide transferase gene under the control of a conditional promoter. We show that ratiometric gas reporting enables the creation of Escherichia coli biosensors that report on acylhomoserine lactone (AHL) autoinducers used for quorum sensing by Gram-negative bacteria. Using these biosensors, we find that an agricultural soil decreases the bioavailable concentration of a long-chain AHL up to 100-fold. We also demonstrate that these biosensors can be used in soil to nondisruptively monitor AHLs synthesized by Rhizobium leguminosarum and degraded by Bacillus thuringiensis. Finally, we show that this new reporting approach can be used in Shewanella oneidensis, a bacterium that lives in sediments.