Comparative analysis of three studies measuring fluorescence from engineered bacterial genetic constructs.
Comparative analysis of three studies measuring fluorescence from engineered bacterial genetic constructs.
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DOI：
10.1371/journal.pone.0252263
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发表时间：
2021
期刊：
影响因子：
3.7
通讯作者：
iGEM Interlab Study Contributors
中科院分区：
文献类型：
--
作者：
Beal J;Baldwin GS;Farny NG;Gershater M;Haddock-Angelli T;Buckley-Taylor R;Dwijayanti A;Kiga D;Lizarazo M;Marken J;de Mora K;Rettberg R;Sanchania V;Selvarajah V;Sison A;Storch M;Workman CT;iGEM Interlab Study Contributors
再现性是合成生物学的一个关键挑战，但再现性的基础只有与它所建立的参考材料一样坚实。在这里，我们重点关注用工程基因构建体转化的细菌的荧光测量的再现性。该比较分析包括三个大型实验室间研究，使用流式细胞术和读板仪、相同的基因构建体和兼容的单位校准协议。在所有三项研究中，我们发现用于酶标仪的校准物具有类似的高精度。我们还发现，流式细胞术结果和两年的读板器结果的荧光测量结果非常一致，平均标准偏差为 1.52 倍，而第三年的读板器结果始终偏移超过一个数量级，平均偏移为 28.9 倍。分析可能的误差源表明这种变化是由于荧光素校准物的制备不正确造成的。这些发现表明，测量工程构建体的荧光具有高度可重复性，但也迫切需要获得用于酶标仪的质量受控的荧光校准物。
Reproducibility is a key challenge of synthetic biology, but the foundation of reproducibility is only as solid as the reference materials it is built upon. Here we focus on the reproducibility of fluorescence measurements from bacteria transformed with engineered genetic constructs. This comparative analysis comprises three large interlaboratory studies using flow cytometry and plate readers, identical genetic constructs, and compatible unit calibration protocols. Across all three studies, we find similarly high precision in the calibrants used for plate readers. We also find that fluorescence measurements agree closely across the flow cytometry results and two years of plate reader results, with an average standard deviation of 1.52-fold, while the third year of plate reader results are consistently shifted by more than an order of magnitude, with an average shift of 28.9-fold. Analyzing possible sources of error indicates this shift is due to incorrect preparation of the fluorescein calibrant. These findings suggest that measuring fluorescence from engineered constructs is highly reproducible, but also that there is a critical need for access to quality controlled fluorescent calibrants for plate readers.
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影响因子：
3.7
作者：
Beal J;Haddock-Angelli T;Gershater M;de Mora K;Lizarazo M;Hollenhorst J;Rettberg R;iGEM Interlab Study Contributors
通讯作者：
iGEM Interlab Study Contributors
影响因子：
3.7
作者：
Beal J;Haddock-Angelli T;Baldwin G;Gershater M;Dwijayanti A;Storch M;de Mora K;Lizarazo M;Rettberg R;with the iGEM Interlab Study Contributors
通讯作者：
with the iGEM Interlab Study Contributors
影响因子：
4
作者：
Milo, Ron
通讯作者：
Milo, Ron
影响因子：
3.7
作者：
Hoffman, Robert A.;Wang, Lili;Nolan, John P.
通讯作者：
Nolan, John P.
影响因子：
4.7
作者：
Roehner, Nicholas;Beal, Jacob;Myers, Chris J.
通讯作者：
Myers, Chris J.