Applicability of Control Materials To Support Gene Promoter Characterization and Expression in Engineered Cells Using Digital PCR.
Applicability of Control Materials To Support Gene Promoter Characterization and Expression in Engineered Cells Using Digital PCR.
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DOI：
10.1021/acs.analchem.1c05134
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发表时间：
2022-04-12
影响因子：
7.4
通讯作者：
Whale, Alexandra S.
中科院分区：
文献类型：
--
作者：
Fernandez-Gonzalez, Ana;Cowen, Simon;Kim, Juhyun;Foy, Carole A.;Jimenez, Jose;Huggett, Jim F.;Whale, Alexandra S.
在工程中使用标准化的组件和过程是设计-建造-测试模型的基础，生物系统的工程也不例外。对组件和验证工程生物系统的方法进行标准化的大量努力正在进行中。本研究开发了一组对照材料，将常用的报告基因GFP和RFP编码为DNA或RNA分子。每个小组最多包含6个样本，两个报告基因之间的拷贝数差异越来越小，差异范围从1到2倍。这些拷贝数差异代表了可能需要测量以验证工程系统的变化幅度。使用数字PCR (dPCR)，我们证明了可以将样本内部和样本之间的基因和基因转录数的变化量化到1.05倍。我们在细菌模型中使用一个简单的基因回路证实了这些发现，以证明dPCR能够精确识别两种转录本在响应启动子刺激时基因表达的微小变化。最后，我们利用我们的发现来强调误差来源，这些误差可能导致生物系统中小比例测量的测量不确定度。总之，控制材料面板的开发和用于测量基因表达微小变化的高精度方法的验证，本研究可以为工程生物学的方法和材料“工具包”做出贡献，以支持当前的标准化工作。
The use of standardized components and processes in engineering underpins the design-build-test model, and the engineering of biological systems is no different. Substantial efforts to standardize both the components and the methods to validate the engineered biological systems is ongoing. This study has developed a panel of control materials encoding the commonly used reporter genes GFP and RFP as DNA or RNA molecules. Each panel contained up to six samples with increasingly small copy number differences between the two reporter genes that ranged from 1- to 2-fold differences. These copy number differences represent the magnitude of changes that may need to be measured to validate an engineered system. Using digital PCR (dPCR), we demonstrated that it is possible to quantify changes in both gene and gene transcript numbers both within and between samples down to 1.05-fold. We corroborated these findings using a simple gene circuit within a bacterial model to demonstrate that dPCR was able to precisely identify small changes in gene expression of two transcripts in response to promoter stimulation. Finally, we used our findings to highlight sources of error that can contributed to the measurement uncertainty in the measurement of small ratios in biological systems. Together, the development of a panel of control materials and validation of a high accuracy method for the measurement of small changes in gene expression, this study can contribute to the engineering biology “toolkit” of methods and materials to support the current standardization efforts.
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DOI：
10.1534/g3.117.300123
发表时间：
2017-10-05
期刊：
G3 (Bethesda, Md.)
影响因子：
--
作者：
Falabella M;Sun L;Barr J;Pena AZ;Kershaw EE;Gingras S;Goncharova EA;Kaufman BA
通讯作者：
Kaufman BA
影响因子：
5.9
作者：
Hecht, Ariel;Filliben, James;Salit, Marc
通讯作者：
Salit, Marc
DOI：
10.3390/bios8010023
发表时间：
2018-03-12
期刊：
Biosensors
影响因子：
--
作者：
Neethirajan S;Ragavan V;Weng X;Chand R
通讯作者：
Chand R
影响因子：
9.3
作者：
Whale, Alexandra S.;Jones, Gerwyn M.;Devonshire, Alison S.
通讯作者：
Devonshire, Alison S.
影响因子：
9.3
作者：
Huggett, Jim F.;Foy, Carole A.;Bustin, Stephen A.
通讯作者：
Bustin, Stephen A.