Transcript Barcoding Illuminates the Expression Level of Synthetic Constructs in E. coli Nissle Residing in the Mammalian Gut
Transcript Barcoding Illuminates the Expression Level of Synthetic Constructs in E. coli Nissle Residing in the Mammalian Gut
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DOI：
10.1021/acssynbio.0c00040
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发表时间：
2020-05-15
影响因子：
4.7
通讯作者：
Dantas, Gautam
中科院分区：
文献类型：
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作者：
Crook, Nathan;Ferreiro, Aura;Dantas, Gautam
强大的工程益生菌疗法的开发需要准确了解肠道中基因构建体的表达。然而，在脊椎动物宿主中测试工程菌株的金钱和道德成本与当前的高通量设计-构建-测试周期不相容。为了能够并行测量多个构建体设计，我们将独特的DNA条形码放置在工程化的转录物中，并通过测序测量条形码丰度。在标准曲线实验中，条形码序列在输入和测量的丰度之间表现出一致的关系，这使得我们能够使用转录物条形码来测量30种表达GFP的大肠杆菌菌株的表达水平。coliNissle平行培养。将该技术应用于培养物和小鼠肠道中，我们发现肠道中的GFP表达通常可以从培养物中的表达水平预测，但几种菌株表现出肠道特异性表达。这项工作建立了实验设计参数和转录物条形码化的优势，以测量许多工程益生菌设计在哺乳动物宿主中的性能。
The development of robust engineered probiotic therapies demands accurate knowledge of genetic construct expression in the gut. However, the monetary and ethical costs of testing engineered strains in vertebrate hosts are incompatible with current high-throughput design-build-test cycles. To enable parallel measurement of multiple construct designs, we placed unique DNA barcodes in engineered transcripts and measured barcode abundances via sequencing. In standard curve experiments, the barcode sequences exhibited consistent relationships between input and measured abundances, which allowed us to use transcript barcoding to measure expression levels of 30 GFP-expressing strains of E. coli Nissle in parallel. Applying this technology in culture and in the mouse gut, we found that GFP expression in the gut could often be predicted from expression levels in culture, but several strains exhibited gut-specific expression. This work establishes the experimental design parameters and advantages of transcript barcoding to measure the performance of many engineered probiotic designs in mammalian hosts.