RNA-aptamers-in-droplets (RAPID) high-throughput screening for secretory phenotypes.
RNA-aptamers-in-droplets (RAPID) high-throughput screening for secretory phenotypes.
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RNA-APTAMER-IN-DROPLETS（快速）分泌表型的高通量筛选。
DOI：
10.1038/s41467-017-00425-7
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发表时间：
2017-08-23
影响因子：
16.6
通讯作者：
Abate AR
中科院分区：
文献类型：
--
作者：
Abatemarco J;Sarhan MF;Wagner JM;Lin JL;Liu L;Hassouneh W;Yuan SF;Alper HS;Abate AR
合成生物学和代谢工程寻求将微生物重新设计成“活的铸造厂”，用于生产高价值化学品。通过“设计-构建-测试”循环范例，可以构建大量基因工程微生物库并测试代谢物的过量生产和分泌。然而，文库生成能力超过了高通量测试和筛选的速度。孔板测定很灵活，但通量有限，而液滴微流控技术具有超高通量，但需要针对每个目标进行定制测定。在这里，我们提出了液滴中RNA适体（RAPID），这种方法极大地扩展了超高通量微流体筛选的通用性。使用适配体，我们将细胞外产物滴度转换为荧光，从而可以对数百万个变体进行超高通量筛选。我们通过将酿酒酵母中酪氨酸的产生和重组蛋白的分泌分别提高高达 28 倍和 3 倍来证明 RAPID 方法。液滴适体为进化微生物提供了合成和分泌增值化学物质的通用方法。筛选基因工程微生物文库中的分泌产物受到可用检测通量的限制。在这里，作者将基于适体的荧光检测与液滴微流体相结合，以实现对旨在增强酪氨酸或链霉亲和素产量的酵母菌株的高通量筛选。
Synthetic biology and metabolic engineering seek to re-engineer microbes into “living foundries” for the production of high value chemicals. Through a “design-build-test” cycle paradigm, massive libraries of genetically engineered microbes can be constructed and tested for metabolite overproduction and secretion. However, library generation capacity outpaces the rate of high-throughput testing and screening. Well plate assays are flexible but with limited throughput, whereas droplet microfluidic techniques are ultrahigh-throughput but require a custom assay for each target. Here we present RNA-aptamers-in-droplets (RAPID), a method that greatly expands the generality of ultrahigh-throughput microfluidic screening. Using aptamers, we transduce extracellular product titer into fluorescence, allowing ultrahigh-throughput screening of millions of variants. We demonstrate the RAPID approach by enhancing production of tyrosine and secretion of a recombinant protein in Saccharomyces cerevisiae by up to 28- and 3-fold, respectively. Aptamers-in-droplets affords a general approach for evolving microbes to synthesize and secrete value-added chemicals. Screening libraries of genetically engineered microbes for secreted products is limited by the available assay throughput. Here the authors combine aptamer-based fluorescent detection with droplet microfluidics to achieve high throughput screening of yeast strains engineered for enhanced tyrosine or streptavidin production.
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影响因子：
--
作者：
Aharoni, A;Amitai, G;Tawfik, DS
通讯作者：
Tawfik, DS
DOI：
10.1073/pnas.1506460112
发表时间：
2015-08-25
影响因子：
11.1
作者：
Huang, Mingtao;Bai, Yunpeng;Nielsen, Jens
通讯作者：
Nielsen, Jens
影响因子：
3.9
作者：
Johnson JR;D'Amore R;Thain SC;Craig T;McCue HV;Hertz-Fowler C;Hall N;Hall AJ
通讯作者：
Hall AJ
影响因子：
16.6
作者：
Colin PY;Kintses B;Gielen F;Miton CM;Fischer G;Mohamed MF;Hyvönen M;Morgavi DP;Janssen DB;Hollfelder F
通讯作者：
Hollfelder F
影响因子：
16.6
作者：
Crook, Nathan;Abatemarco, Joseph;Sun, Jie;Wagner, James M.;Schmitz, Alexander;Alper, Hal S.
通讯作者：
Alper, Hal S.